Methods and compositions to alter the cell surface expression of phosphatidylserine and other clot-promoting plasma membrane phospholipids

ABSTRACT

A protein preparation that mediates Ca +2  transbilayer movement of phospholipid is disclosed. Additionally, a modified or mutated protein preparation, wherein the protein has a reduced ability to mediate transbilayer movement, is disclosed. In a preferred form of the invention, the protein has been modified such that post-translational modification can no longer occur.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of Ser. No.08/790,186, which claims priority to Serial No. 60/015,385. Both ofthese applications are incorporated by reference as if fully set forthherein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] United States Government may have commercial rights under GrantR01 HL36946 from Heart, Lung, & Blood Institute, National Institutes ofHealth.

BACKGROUND OF THE INVENTION

[0003] The exposure of phosphatidylserine (PS) and otheraminophospholipids (aminoPL) on the surface of activated or injuredblood cells and endothelium is thought to play a key role in theinitiation and regulation of blood coagulation. De novo surface exposureof aminophospholipids has also been implicated in the activation of bothcomplement and coagulation systems after tissue injury, and in removalof injured or apoptotic cells by the reticuloendothelial system.Although migration of these phospholipids (PL)from inner-to-outer plasmamembrane leaflets is known to be triggered by elevated intracellular[Ca²⁺] ([Ca²⁺]_(c)) and to be associated with vesicular blebbing of thecell surface, little is known about the cellular constituents thatparticipate in this process.

[0004] As described in Ser. No. 08/790,186, cell surface PS has a rolein coagulation, programmed cell death and clearance by thereticuloendothelial system. Ser. No. 08/790,186 also describesregulation of the transmembrane distribution of PS, the role of calciumin the collapse of phospholipid asymmetry, and the role PL translocationin Scott Syndrome.

[0005] Bassé, et al. and Stout, et al. recently reported thepurification and preliminary characterization of an integral RBCmembrane protein that, when reconstituted in liposomes, mediates aCa²⁺-dependent transbilayer movement of PL mimicking plasma membrane PLreorganization evoked upon elevation of [Ca²⁺]_(c) (F. Bassé, et al., J.Biol. Chem. 271:17205-17210, 1996; J. G. Stout, et al., J. Clin. Invest.99:2232-2238, 1997). Evidence that a protein of similar function mustalso be present in platelets was recently reported by Comfurius, et al.(P. Comfurius, et al., Biochemistry 35:7631-7634, 1996).

[0006] Needed in the art is a method for modulating the activity ofphospholipid scramblase within a cell, organ or tissue in which onewishes either to reduce the potential for thrombosis, clot formation, orcell clearance (by decreasing cellular PL scramblase expression oractivity) or to promote hemostasis or cell clearance (by increasingcellular PL scramblase expression or activity).

BRIEF SUMMARY OF THE INVENTION

[0007] The present invention relates to the creation and use ofantithrombotic and thrombostatic reagents that rely on the properties ofa protein preparation that mediates Ca²⁺-dependent transbilayer movementof membrane phospholipids.

[0008] In one embodiment, the present invention is a preparation of aplasma membrane phospholipid scramblase (“PL scramblase”). Preferably,the protein is approximately 35-37 kD as measured on a 12.5%SDS-polyacrylamide gel under reducing conditions. In a most preferredform of this invention, the preparation is a human or a mouse PLscramblase.

[0009] In one preferred embodiment of the present invention, the PLscramblase comprises SEQ ID NO:2, representing human PL scramblase,possibly with conservative or functionally equivalent substitutions.

[0010] In the most preferred embodiment of the present invention, the PLscramblase, preferably comprising SEQ ID NO:2, has been modified by theaction of mammalian cellular enzymes to covalently incorporatephosphorous at one or more Thr, Ser, or Tyr residues or a fatty acid,preferably palmitate, at a cysteine residue.

[0011] The present invention is also a preparation of a murine cellprotein, wherein the protein is a plasma membrane phospholipidscramblase, preferably wherein the protein is approximately 35 kD asmeasured on a 12.5% SDS-polyacrylamide gel under reducing conditions.

[0012] In one preferred embodiment of the present invention, the murinePL scramblase comprises SEQ ID NO:4, possibly with conservative orfunctionally equivalent substitutions.

[0013] In the most preferred embodiment of the present invention, themurine PL scramblase comprising SEQ ID NO:4 has been modified by theaction of mammalian cellular enzymes to covalently incorporatephosphorous one or more Thr, Ser, or Tyr residues, and a fatty acid,preferably palmitate, at a cysteine residue.

[0014] The present invention is also a DNA sequence encoding the PLscramblase. Preferably, this DNA sequence comprises SEQ ID NO:1. Mostpreferably, this DNA sequence comprises residues 211-1164 of SEQ IDNO:1.

[0015] The present invention is also a DNA sequence encoding the murinePL scramblase. Preferably, this DNA sequence comprises SEQ ID NO:3. Mostpreferably, the DNA sequence comprises residues 192-1112 of SEQ ID NO:3.

[0016] In another embodiment, the present invention is a method ofpreventing the surface exposure of plasma membrane phospholipids andreducing the procoagulant properties of a cell by delivering to the cella mutant phospholipid scramblase. This scramblase is preferably mutatedat a site of post-translational modification, most preferably the siteis selected from the group consisting of Asp273-Asp284, Thr161 andCys297 of human PL scramblase SEQ ID NO:2 or the corresponding conservedresidues in mouse or other mammalian PL scramblase.

[0017] In one embodiment, a gene construct encoding a mutantphospholipid scramblase is delivered to the cell. In an alternativeembodiment, the mutant protein itself is delivered.

[0018] The present invention is also an inhibitor of the PL scramblaseactivity of PL scramblase. This inhibitor may be an antisense nucleotidederived from the DNA sequence of PL scramblase. In another embodiment,the inhibitor is a peptide sequence that is a competitive inhibitor ofPL scramblase activity. In another embodiment, the inhibitor is anantibody, preferably a monoclonal antibody, raised against PLscramblase.

[0019] In another embodiment, the inhibitor works by modifying orinhibiting the post-translational modifications of the PL scramblasethat are disclosed below in the Examples. For example, analysis of theprimary PL scramblase sequence reveals a potential site ofphosphorylation by protein kinase C or other cellular kinase (Thr161), apotential site for acylation by fatty acid (Cys297), and a potentialbinding site for Ca²⁺ ion provided by an EF-hand-like loop spanningresidues Asp273-Asp284. These residues and motifs are conserved in themouse PL scramblase.

[0020] In one embodiment, the inhibitor is a compound that preventsthioacylation of the protein.

[0021] In another embodiment, a mutant phospholipid scramblase isprovided in which cysteine residues, preferably Cys297 of SEQ ID NO:2(or the equivalent residue in the conserved region of another PLscramblase), have been replaced by alanine or other non-conservativesubstitution.

[0022] The present invention is also a method for preventing the surfaceexposure of plasma membrane phosphatidylserine, phosphatidylethanolamineand cardiolipin on the surface of in vitro stored leukocytes,lymphocytes, platelets or red blood cells. This method comprises thesteps of adding an inhibitor of PL scramblase activity to the storedblood cells.

[0023] The present invention is also a method for prolonging survival oftransplanted organs comprising the step of adding an inhibitor of PLscramblase activity to an organ perfusate during in vitro organ storage.The present invention is also a method for prolonging the survival oftransplanted cells, tissues, and organs by genetically engineering thecells to be transplanted so as to alter their expression of plasmamembrane PL scramblase in order to reduce exposure of PS and otherthrombogenic phospholipids at the plasma membrane surface, therebyreducing the risk of infarction due to fibrin clot formation.

[0024] The present invention is also a method for prolonging the in vivosurvival of circulating blood cells (erythrocyte, platelets, lymphocyte,PMN's, and monocytes) comprising the step of preventing surface exposureof plasma membrane phosphatidylserine on the surface of the cells byexposing the blood cells to an inhibitor of PL scramblase activity.

[0025] The present invention is also a method for preventing theprocoagulant activities of erythrocytes in sickle cell diseasecomprising the step of inhibiting erythrocyte PL scramblase in a sicklecell patient.

[0026] The present invention is also a method for treating autoimmuneand inflammatory diseases comprising the step of treating a patient withan inhibitor of the PL scramblase activity of PL scramblase.

[0027] The present invention is also a method for diagnosing individualswith reduced or elevated capacity for platelet-promoted orerythrocyte-promoted fibrin clot activity comprising the step ofquantifying the cellular expression of PL scramblase. This quantitationmay take the form of immunoblotting using an antibody to PL scramblase,an ELISA assay using an antibody to PL scramblase, flow cytometricanalysis of the binding of monoclonal antibody reactive against thepredicted extracellular domain of PL scramblase (residues Ser310-Tryp318of sequence disclosed in SEQ ID NO:2 or the equivalent residue in theconserved region of another PL scramblase) or using oligonucleotidesderived from PL scramblase cDNA and the polymerase chain reaction. Inone method of the present invention, the quantitation is performed byisolating PL scramblase from a patient blood sample, measuring theamount of PL scramblase isolated and comparing the measurement with acontrol sample. The measurement may be by isolating PL scramblase from apatient blood sample and measuring via densitometry the amount of PLscramblase protein electrophoresed in a stained electrophoretic gel.

[0028] It is an object of the present invention to provide a preparationof a PL scramblase.

[0029] It is another object of the present invention to geneticallyalter the level of expression of PL scramblase by delivery of cDNArepresenting sense or antisense nucleotide sequence ligated into asuitable mammalian expression vector.

[0030] It is another object of the present invention to provide aninhibitor of PL scramblase PL scramblase activity.

[0031] It is another object of the present invention to provide anantithrombotic agent.

[0032] It is another object of the present invention to create cells,tissue, and organs for transplantation that have increased potential forsurvival and reduced potential for causing fibrin clot formation andvascular thrombosis when grafted into a recipient host.

[0033] It is another object of the present invention to create ananimal, preferably a mouse or pig, that has been genetically engineeredso that the PL scramblase gene is not expressed.

[0034] Other objects, advantages and features of the present inventionwill become apparent after one of skill in the art reviews thespecification, claims and drawings herein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0035]FIG. 1A is a comparison of the cDNA and deduced amino acidsequence of human PL scramblase (SEQ ID NOs:1 and 2).

[0036]FIG. 1B is a comparison of the cDNA and deduced amino acidsequence of murine PL scramblase (SEQ ID NOs:3 and 4).

[0037]FIG. 2 is a bar graph illustrating immunoprecipitation oferythrocyte PL scramblase.

[0038]FIG. 3 is a graph of an activity assay of recombinant PLscramblase.

[0039]FIG. 4 is an immunoblot of PL scramblase in human erythrocytes andplatelets.

[0040]FIG. 5 is a comparison of protein sequences of mouse and human PLscramblase (SEQ ID NOs:2 and 4).

[0041]FIG. 6 is a bar graph of PL scramblase activity as a function ofmutational analysis of a putative EF hand loop motif contained in humanPL scramblase.

[0042]FIG. 7 graphs the Ca²⁺ dependence of mutant human PL scramblase.

[0043]FIG. 8 is a Western blot analysis of PL scramblase protein andcorresponding functional assay of PL scramblase activity in varioushuman cell lines.

[0044]FIG. 9A and B are fluorescence micrographs of GFP-PL scramblase intransformed Raji cells. FIG. 9A depicts fluorescence of cells expressingGFP; FIG. 9B depicts cells transfected with pEGFP-C2-PL scramblaseplasmid and expressing GFP-PL scramblase fusion protein.

[0045]FIG. 10 is a graph showing that the level of expression of PLscramblase determines plasma membrane sensitivity to intracellularcalcium.

[0046]FIG. 11 is a bar graph illustrating inactivation of PL scramblaseby hydroxylamine.

DETAILED DESCRIPTION OF THE INVENTION

[0047] In the description of the invention presented herein, Applicantsspecifically refer to numerical residue positions in both the PLscramblase amino acid and nucleotide sequence. These reference numbersrefer to the residue position in the amino acid or nucleotide sequencelisted in the Sequence Listing. When Applicants use these referencenumbers to describe a proposed mutated PL scramblase or nucleic acid,Applicants mean for these reference numbers to refer to the comparableor equivalent residue in that protein or amino acid. For example (seeFIG. 5), threonine 161 in the human protein sequence (SEQ ID NO:2) isequivalent to threonine 159 in the mouse protein sequence (SEQ ID NO:4).Although these residues have different reference numbers, they areequivalent residues in the two sequences based upon conserved homologyin the aligned sequences. One may determine what a “equivalent residue”is in an unknown PL scramblase sequence by deducing the highestprobability alignment to the human sequence using BLAST, FASTA or othersequence alignment tool commonly known to those skilled in the art.

[0048] 1. Protein Preparations and Nucleic Acid Sequences

[0049] The Examples below disclose the purification and preliminarycharacterization of an integral RBC membrane protein that, whenreconstituted in liposomes, mediates a Ca²⁺-dependent transbilayermovement of PL mimicking plasma membrane PL reorganization evoked uponelevation of [Ca^(2+]) _(c). Based on internal peptide sequence obtainedfrom the purified erythrocyte PL scramblase protein, we cloned the cDNA(SEQ ID NO:1) encoding this protein from a human K-562 erythroleukemiclibrary (Q. Zhou, et al., J. Biol. Chem. 272:18240-18244, 1997). Thededuced human PL scramblase protein (SEQ ID NO:2) is a single chainpolypeptide of 318 amino acids with molecular weight of 35.1 kD andcalculated isoelectric point of 4.9. It is predicted to be a type 2membrane protein with a single transmembrane domain near the carboxylterminus (residues Ala291-Gly309), a short exoplasmic carboxyl terminalpeptide (residues Ser310-Trp318) with the remaining polypeptide(residues Metl-Lys290) in the cytosol.

[0050] The present invention involves the purification andcharacterization of this approximately 35-37 kD membrane protein thatpromotes a Ca² ⁺-dependent transbilayer redistribution of membranephospholipids including PS and PC, with properties similar to the PLscramblase activity that is evoked upon elevation of Ca²⁺ in the cytosolof erythrocytes and other cells. We have named this membrane protein“37.” We mean for “P37” to be synonymous with “phospholipid scramblase”or “PL scramblase” and refer to these names interchangeably throughoutthe text. By “phospholipid scramblase” or “PL scramblase activity,” wemean the Ca²⁺ dependent transbilayer movement of plasma membranephospholipid.

[0051] In one embodiment, the present invention is a protein preparationof PL scramblase. In preferred embodiments of the present invention, thepreparation is of either human or mouse PL scramblase.

[0052] If one desires the human PL scramblase, preferably, the proteincomprises residues 1-318 of SEQ ID NO:2. More preferably, the PLscramblase comprises residues 85-307 of SEQ ID NO:2, representing onlythe most highly conserved residues of the human PL scramblase whenaligned against murine PL Scramblase (see FIG. 5).

[0053] If one desires the mouse PL scramblase, preferably, the proteincomprises residues 1-307 of SEQ ID NO:4. More preferably, the PLscramblase comprises residues 83-307 of SEQ ID NO:4, representing onlythe most highly conserved residues of the mouse PL scramblase whenaligned against human PL Scramblase (see FIG. 5).

[0054] In another embodiment, the protein comprises conservativesubstitutions or functionally equivalent residues of the residuesdescribed in the paragraph above. By “functionally equivalent” we meanthat the equivalent residues do not inhibit or disrupt the activity ofthe PL scramblase preparation. A protein with “equivalent” substitutionswould have at least a 10%, preferably 50%, activity of a native PLscramblase preparation.

[0055] The Examples below demonstrate one method of isolating PLscramblase from human erythrocytes. After examination of thespecification below, other methods of protein isolation will becomeapparent to one of skill in the art. The Examples below also describe anassay for the measurement of PL scramblase activity. A suitablepreparation of the present invention would have a PL scramblase activityof at least 10% that of the preparation described below in the Examples.Preferably, the activity would be at least 50% that of the Examplesdescribed below.

[0056] We specifically envision that one may wish to isolate the PLscramblase from a variety of mammalian sources including human, mouse,pig, or other mammal.

[0057] In one embodiment of the invention, the PL scramblase is isolatedfrom erythrocyte membranes. In another embodiment, the protein isisolated from one or more body tissues including, spleen, skin, lung orother organ. In another embodiment, the protein is produced by bacteriacells, such as E. coli cells, insect cells, or yeast, preferably invitro cultures that are transfected with plasmid or viral vectorscontaining cDNA sequences identified at SEQ ID NOs:1 or 3 in the correctreading frame. The vector can be chosen from among protein expressionvectors known to those skilled in the art. Preferable viral vectorsinclude retrovirus, adenovirus, and baculovirus vectors.

[0058] The present invention is also a recombinant DNA sequence encodingPL scramblase. A preferable DNA sequence encoding PL scramblase wouldcomprise the residues of SEQ ID NO:1 (human sequence) or SEQ ID NO:3(mouse sequence). A more preferable DNA sequence encoding PL scramblasewould comprise the nucleic acids 211-1164 of SEQ ID NO:1 or 192-1112 ofSEQ ID NO:3. The most preferred DNA sequence encoding PL scramblasewould comprise the nucleic acids 463-1137 of SEQ ID NO:1 or 438-1112 ofSEQ ID NO:3 One of skill in the art of molecular biology would know howto obtain other DNA sequences encoding the PL scramblase. For example,one might sequence PL scramblase directly via standard proteinsequencing techniques. The peptide sequence could be analyzed to provideoligonucleotide probes for a human cDNA leukocyte library. (One suchcDNA library is available from Invitrogen in a pCDNA3 vector.)

[0059] By use of probes obtained from these and other the PLscramblases, one would then be able to isolate other cDNA clonesencoding the entire PL scramblase protein sequence from a species orcell culture of interest. SEQ ID NO:1 contains the entire open readingframe encoding human PL scramblase as well as flanking residues of 5′and 3′ untranslated sequence. The full-length translation of SEQ ID NO:1is identified as SEQ ID NO:2. In the cDNA, this translated sequencewould normally be followed by the appropriate stop codon.

[0060] Based on the nucleotide sequence of human PL scramblase, theExamples below disclose a full-length cDNA for murine PL scramblase froma mouse fibroblast cDNA library (CLONETECH). The resulting cDNA (SEQ IDNO:3) predicts an open reading frame encoding a 307 residue polypeptide(molecular weight 33.9 kDa; calculated pI=4.9) (SEQ ID NO:4). Analysisof the murine PL scramblase protein revealed that it had virtually thesame apparent affinity for Ca²⁺ and the same activity in promotingtransbilayer movement of phospholipids as exhibited by the recombinanthuman protein.

[0061] Alignment of the murine and human PL scramblase proteins reveals65% overall identity of sequence, with the most divergent sequence foundin the amino terminal portion of the protein. The murine carboxylterminus is truncated, and does not include the predicted exoplasmicdomain found in the carboxyl terminus of human PL scramblase. Thissuggests that residues Ser310-Trp318 in human PL scramblase do notcontribute to its function. By contrast, segments of human PL scramblasepolypeptide that are implicated to participate in its phospholipidmobilizing function (detailed below), are highly-conserved in the mouseprotein. These structural motifs that are conserved in both human andmouse PL scramblase include: a single inside-outside transmembranedomain for membrane attachment (human residues Ala291-Gly309); apotential site of phosphorylation by protein kinase C or other cellularkinase (Thr161 in human); a potential site of thiol-acylation withpalmitic acid (Cys297 in human); and a potential binding site for Ca² ⁺ion (residues Asp273-Asp284 in human). Based on the best fit alignmentof the human and mouse protein sequences reported as SEQ ID NO:2 and SEQID NO:4, we deduce that the highly-conserved portions of thepolypeptide, representing residues 85-309 of SEQ ID NO:2 of human PLscramblase and residues 83-307 of SEQ ID NO:4 (i.e., the equivalentresidues of mouse PL scramblase) contains the portion of the proteinrequired for PL scramblase activity.

[0062] The present invention is also a preparation of a modified ormutated PL scramblase wherein the PL scramblase has a reduced ability tomediate transbilayer movement of lipids. By “reduced activity” we meanthat the modified scramblase has less than 10% of the activity of thewild-type scramblase. Preferably, this activity is measured by the Ca²⁺dependent movement of fluorescent phospholipids in reconstitutedproteoliposomes (see Examples 1 and 2). More preferably, this activityis measured by the intracellular Ca²⁺-dependent movement of PS to thecell surface in cells treated or transfected so as to express a modifiedor mutated PL scramblase (see Example 3).

[0063] Preferably, the protein is modified such that it is no longerpost-translationally modified, as described above. Most preferably, themodification occurs at amino acid residue Thr161, Cys297, orAsp273-Asp284.

[0064] The present invention is also a recombinant nucleic acid encodinga modified or mutated scramblase.

[0065] 2. Modulators of PL Scramblase Activity.

[0066] The present invention is also a modulator, either an inhibitor orenhancer, of the PL scramblase activity of PL scramblase. Theinformation below in the Examples demonstrates a new understanding ofthe post-translational modification of PL scramblase that may be used todesign methods of modulating PL scramblase activity and mutated PLscramblases with modified scramblase activity. For example, analysis ofthe primary PL scramblase sequence reveals a potential site ofphosphorylation by protein kinase C or other cellular kinase (Thr161), apotential site for acylation by fatty acid (Cys297), and a potentialbinding site for Ca²⁺ ion provided by an EF-hand-like loop spanningresidues Asp273-Asp284. Knowledge of these post-translationalmodifications allows one to design specific inhibitors or modulators ofthe PL scramblase activity.

[0067] Therefore, as elaborated below in sections A, B and C, thepresent invention is a method of modulating PL scramblase activity bydisrupting specific post-translational modifications. In one embodiment,the method comprises exposing a PL scramblase molecule to apost-translational modification inhibitor and, thus, reducing PLscramblase activity. This method will be useful in a variety ofapplications where reduction of the rate of clearance of a cell from thebody or a reduction in clot promoting and procoagulant activities of acell is desired. Among the post translational modification predicted toalter the activity of PL scramblase include insertion of the proteininto phospholipid membranes, phosphorylation of the polypeptide by anintracellular protein kinase at one or more Tyr, Thr, or Ser residues,the addition of palmitate or other fatty acid by thioacylation throughformation of a thioester bond at one or more Cys residues in thecytoplasmic or transmembrane domains of the protein, the binding of oneor more metal ions to the protein, the aggregation of the protein withitself or one or more cofactors, proteolytic degradation of the proteinby one or more cytoplasmic proteases including by example calpains orcaspases.

[0068] In another embodiment, the method comprises creating a geneconstruct encoding a modified PL scramblase. The scramblase will bemodified at the site of the post-translational modification describedabove. This modified gene may be used to transfect cells that one wishesto display a reduction of clot promoting or procoagulant activities orto prolong the survival of the cell in the body.

[0069] Sections A, B and C below describe specific residues that one maywish to mutate. One of skill in the art would be aware of generalmolecular biological techniques that would enable one to acquire a PLscramblase gene, create the appropriate mutation, create the appropriategenetic construct, and transform the desired cell line.

[0070] In another embodiment, the inhibitor is an antisense nucleotidederived from the DNA sequence encoding PL scramblase. One of skill inthe art would know how to create such an antisense nucleotide from thecDNA sequence of PL scramblase.

[0071] In another embodiment, the inhibitor is an antibody, preferably amonoclonal antibody, raised against PL scramblase. One of skill in theart would know how to make an antibody preparations from the purifiedprotein preparation described below.

[0072] A. Cysteine Thioester

[0073] Our discovery of the presence of a conserved site for Cysthiol-acylation in the transmembrane domains of human and mouse PLscramblase (Cys297 in human) suggests that PL scramblase polypeptide ispost-translationally modified by the attachment of fatty acid, and thatthis thiol-acylation is required for normal expression of its biologicalactivity. Attachment of fatty acid (predominantly palmitic acid) byacylation of the cysteinyl thiol residue has been shown to regulate thebiological activity of a variety of cellular proteins (G. Milligan, etal., Trends Biochem. Sci. 20:181-185, 1995; M. J. Schlesinger, et al.,In: Lipid Modification of Proteins, pp. 1-19, CRC Press, Boca Raton,Fla., 1993).

[0074] One embodiment of the present invention is a method to preventegress of PS and other clot-promoting and procoagulant phospholipids tocell surfaces by preventing or reversing the acylation of cysteines inplasma membrane PL scramblase protein. In one embodiment of the presentinvention, one would create a mutated scramblase, wherein cysteine 297(or the equivalent residue in the conserved region of another PLscramblase) is no longer available for post-translational modification.This may be by substituting the cysteine with a alanine, serine oranother non-functionally equivalent amino acid residue. The cDNAencoding this mutated scramblase may be placed in a vector expressionsystem and used to transfect cells of interest. We envision that themutated scramblase will out-compete native scramblase and, thus, reducescramblase activity.

[0075] In another embodiment, the method preferably comprises the stepsof exposing a PL scramblase to a thiolacylation inhibitor and inhibitingPL scramblase activity. Applicants note that the thiolacylationinhibitor could either inhibit thiolacylation directly, block the siteof thiolacylation, or hydrolyze pre-existing thioester-linked fattyacids attached to the protein. Applicants specifically envision that thethiolacylation may be prevented by compounds selected from the class ofspecific antibodies against the protein that react at the site ofthioacylation, thiol-reactive compounds that covalently modify cysteineresidues (including by example N-ethyl maleimide, iodoacetamide, orpyridyldithioethylamine), an inhibitor of enzyme acyltransferasesincluding by example an esterase inhibitor chosen from among carbamates(e.g., physostigmine)and organophosphorus (e.g.,diisopropylfluorophosphate) compounds that are reactive at the activesite of such enzymes. Such a method will be useful for many of theobjects described above, such as treating cells, tissues, and organs fortransplantation to reduce potential for causing fiber and clot formationin vascular thrombosis when grafted into a recipient host. The methodcould also be used to provide an antithrombotic therapeutic effect. Themethod could also be used to increase in vivo survival of the transfusedor transplanted cell by suppressing exposure of PS or otheraminophospholipids at the cell surface.

[0076] B. Peptide Residues Involved in Binding Ca²⁺.

[0077] The phospholipid transport function of PL scramblase is activatedby Ca²⁺ with apparent affinity of 50-100 micromolar, implying arelatively low affinity binding site for the calcium ion within thepolypeptide (Q. Zhou, et al., J. Biol. Chem. 272:18240-18244, 1997; J.G. Stout, et al., J. Clin. Invest. 99:2232-2238, 1997; F. Bassé, et al.,J. Biol. Chem. 271:17205-17210, 1996). The deduced protein sequence ofmouse and human PL scramblase reveals an extensive segment of highlyconserved sequence extending through residue Glu306 (or the equivalentresidue in the conserved region of another PL scramblase). The predictedsecondary structure through this portion of the protein reveals that itcontains two short alpha-helical segments near the C-terminus that areseparated by a 12-residue acidic loop. In both proteins (human andmouse), the C-terminal alpha helix represents a predicted transmembranesegment with a strongly-preferred inside-to-outside orientation, whereassequence contained within the adjacent 12-residue acidic loop conformsin-part to a consensus sequence that is characteristic of an EF-handCa²⁺-binding motif (S. Nakayama, et al., Annu. Rev. Biophys. Biomol.Struct. 23:473-507, 1994). In this motif, residues in positions 1, 3, 5,7, 9 and 12 contribute to octahedral coordination of the Ca²⁺ ion, withthe residues in position 1 [Asp], 3 [Asp, Asn, or Ser] and 12 [Asp orGlu] being those most highly conserved. In order to gain insight intowhether this segment of the protein might be directly involved in theCa²⁺-dependent reorganization of membrane PL mediated by PL scramblase,we expressed mutant human PL scramblase with Asp→Ala substitutions atpositions corresponding to residues 1 (i.e., Asp273), 3 (i.e., Asp275),and 12 (i.e., Asp284) of this putative 12 residue EF-hand loop. Whereasmutations in positions 1 or 12 lead to a partial loss of function,mutation in position 3 resulted in complete inactivation of theCa²⁺-dependent response. The partial loss in activity of PL scramblasewith mutations in positions 1, 3, or 12 was accompanied by a significantreduction in apparent avidity for Ca²⁺. These data identify the segmentof human PL scramblase between Asp273-Asp284 as containing the essentialbinding site for Ca²⁺ and suggest that the activity of this protein canbe selectively inhibited by blocking access of Ca²⁺ to this segment ofthe polypeptide or by modifying residues contained in this segment ofthe polypeptide.

[0078] Therefore, in another embodiment, the present invention is amethod for inhibiting the clot-promoting and procoagulant activity ofthe plasma membrane by preventing the binding of intracellular Ca²⁺ tothe PL scramblase polypeptide. In one embodiment, the method comprisesconstructing a mutant PL scramblase, wherein the PL scramblase ismutated between Asp273 and Asp284 so that the scramblase does not bindCa²⁺ with the same affinity as PL scramblase. This mutant gene may beused, by methods known to one of skill in the art, to transfect a cellor cell line in which one wishes to modulate the clot promoting orprocoagulant properties. We envision that the mutated PL scramblase willout-compete native PL scramblase and thus modulate, preferably reduce,PL scramblase activity in the cell or cell line.

[0079] In another embodiment, this method preferably comprises the stepsof exposing a PL scramblase to a calcium binding inhibitor and reducingPL scramblase activity. Applicants specifically envision that thisinhibitor may be either a direct inhibitor of calcium binding or wouldbind to the residues described above and block calcium binding. Thismethod, as above, would be useful to prepare cells, tissues and organsfor transplantation and as a therapeutic antithrombotic.

[0080] C. Modification of PL Scramblase Through Phosphorylation byCellular Protein Kinases

[0081] As another embodiment, the present invention includes a method toprevent phosphorylation of PL scramblase at one or more Tyr, Thr or Serresidues by inhibiting the action of intracellular Tyr or Ser/Thrkinases. The amino acid sequence of PL scramblase reveals potentialsites of phosphorylation at Tyr, Thr, or Ser residues by cellularprotein kinases, including the conserved motif Thr161-Leu162-Arg163 ofhuman SEQ ID NO:2 (corresponding to Thr159-Leu160-Arg161 of mouse SEQ IDNO:4) predicting phosphorylation by protein kinase C (Q. Zhou, et al.,supra, 1997 and FIG. 5). Protein phosphorylation by one or more cellularprotein kinases is known to regulate many aspects of cell function,which can include both the activation and inactivation of specificenzyme activities. In the specific case of PL scramblase, depletion ofcellular ATP has been shown to inhibit surface exposure ofphosphatidylserine on erythrocytes exposed to elevated [Ca²⁺]_(c) D. W.Martin, et al., J. Biol. Chem. 270:10468-10474, 1995). In combinationwith our discovery of conserved motifs for phosphorylation of PLscramblase, we propose that normal PL scramblase activity requiresconstitutive phosphorylation of the polypeptide. Such phosphorylationsinvariable occur at one or more tyrosines (ie., by tyrosine proteinkinases) or at one or more serines or threonines (i.e. by Ser/Thrprotein kinases). The specific sites of phosphorylation Within a givenprotein are readily identified by finding conserved sequence motifspredictive of phosphorylation, and confirmed using methods known tothose skilled in the art. Such methods include metabolically labelingthe cellular proteins with ³²P, purifying the protein using specificantibody, and identifying the specific residues of polypeptide sequencethat contain covalently bound ³²P, standardly performed by trypticcleavage of the isolated protein, HPLC separation of resulting peptides,and identification of the phosphorylated residues by either Edmandegradation or mass spectroscopic analysis.

[0082] As another embodiment, the Examples below specifically discloseThr161 (or the equivalent residue in the conserved region of another PLscramblase) as a single predicted site of phosphorylation by proteinkinase C. Disruption of this phosphorylation or modification of theparticular residues involved would modify PL scramblase activity.Therefore, the present invention is a method for altering theprocoagulant activity of the plasma membrane by preventingphosphorylation by protein kinases, such as protein kinase C. In oneembodiment, the method comprises creating a mutant PL scramblase,wherein the mutant PL scramblase does not contain a site capable ofphosphorylation by cellular protein kinase. Preferably, the mutant PLscramblase is mutated at Thr161. One then creates a gene constructcapable of expressing the mutated PL scramblase and transfects a cell orcell line of interest. In this manner, one introduces a mutant PLscramblase into the cell or cell line and out competes native orwild-type PL scramblase, thus altering the PL scramblase activity of thecell. In another embodiment, the method preferably comprises the stepsof exposing a PL scramblase to a phosphorylation inhibitor and thusaltering PL scramblase activity. Applicants specifically envision thatthis inhibitor may be either a general phosphorylation inhibitor or mayspecifically block phosphorylation at Thr161. As described above, themethod would be useful to prepare cells tissues and organs fortransplantation and as a therapeutic antithrombotic.

[0083] 3. Expression of PC Scramblase in Human Platelet, HumanEndothelium and other Cell Types

[0084] Our results described below in the Examples confirm that thelevel of expression of plasma membrane PL scramblase can determine theextent to which PS is mobilized to the cell surface upon elevation of[Ca²⁺]_(c), and suggest that this protein normally functions to mediatethe redistribution of plasma membrane phospholipids in response to theentry of calcium into the cytosol.

[0085] These data provide the first experimental demonstration that thecellular potential to mobilize PS and other procoagulantaminophospolipids from plasma membrane inner leaflet to the cellsurface—and thereby expose binding sites for factor Va or other plasmacoagulation factor—can be manipulated by selectively altering the levelof expression of a particular cellular protein, either through directtransfection with the PL scramblase cDNA, by another interventionaffecting either total cellular expression of PL scramblase protein or apost-translational modification of the PL scramblase polypeptide that isessential for its PS mobilizing function in the plasma membrane.

[0086] In one embodiment, the present invention is a method of eitherincreasing or decreasing the clot-promoting and procoagulant propertiesof cell surfaces by either increasing or decreasing the level ofcellular expression PL scramblase mRNA and protein.

[0087] 4. Other Embodiments

[0088] The present invention is also a method for preventing the surfaceexposure of plasma membrane phospholipids, such as phosphatidylserine,phosphatidylethanolamine and cardiolipin, on the surface of in vitrostored blood cells (including, platelets, red blood cells, lymphocytes,or leukocytes) by adding an inhibitor or modulator of the PL scramblaseactivity of PL scramblase to the stored cells.

[0089] The present invention is also a method for prolonging survival oftransplanted organs and grafts comprising the step of adding aninhibitor of PL scramblase PL scramblase activity to an organ perfusateduring in vitro organ storage. The present invention is also a methodfor prolonging the survival of transplanted cells, tissues, and organsby genetically engineering the cells to be transplanted so as to altertheir expression of plasma membrane PL scramblase in order to reduceexposure of PS and other thrombogenic phospholipids at the plasmamembrane surface, thereby reducing the risk of infarction due to fibrinclot formation.

[0090] Therefore, in one embodiment, the present invention is agenetically engineered cell for transplantation into a human or animalwherein the cell has a lowered PL scramblase expression. Preferably, thecell expresses no PL scramblase. Preferably, this cell comprises anucleotide molecule which is expressed by the cell and which codes forprotein inhibiting the activity of PL scramblase. In another preferableembodiment, the promotor of the PL scramblase gene is altered to eitherincrease or decrease the expression of the gene. One of skill in the artof molecular biology would envision methods to create these alteredcells.

[0091] The present invention is also an animal, such as a mouse or pig,that has been genetically manipulated to “knock out” PL scramblaseexpression. Such an animal may be created by many variations oftechniques known to one of skill in the art. Most preferably, one woulddelete by homologous recombination one or more exons of the PLscramblase gene within the chromosomal DNA of the appropriate embryonicstem cell of mouse, pig, or other animal. In the mouse, those exons mostfavored for deletion by homologous recombination are those that includepart or all of DNA sequence between residues 438-1112 of SEQ ID NO:3,representing the conserved portion of the cDNA open reading framerequired for expression of functional PL scramblase protein. Thoseembryonic stem cells showing the PL scramblase gene deletion aresurgically implanted within the uterus at the appropriate time in theestrus cycle. The resulting animals carrying the defective gene are thenbred to homozygosity for the PL scramblase gene deletion defect.

[0092] Preferably, the engineered cell is selected from the groupconsisting of endothelial cells, fibroblasts, epithelial cells, skeletalcells, cardiac and smooth muscle cells, hepatocytes, pancreatic isletcells, bone marrow cells, astrocytes, and Schwann cells. The presentinvention is also a prosthesis for implantation in an animal or humanhaving the genetically engineered cells attached thereto. In oneembodiment, the prosthesis is a vascular graft.

[0093] The present invention is also a method for prolonging the in vivosurvival of circulating blood cells comprising the step of preventingsurface exposure of plasma membrane phosphatidylserine on thecirculating blood cells by inhibiting the function of plasma membrane PLscramblase. One may also wish to prevent the procoagulant properties oferythrocytes in sickle cell disease by inhibiting erythrocyte PLscramblase in a sickle cell patient.

[0094] The present invention is also a method for treating autoimmuneand inflammatory diseases, such as disseminated intravascularcoagulation, vascular thrombosis, fibrin generation duringcardiopulmonary bypass procedures, rheumatoid arthritis, systemic lupuserythematosus, thrombotic thrombocytopenic purpura, heparin-associatedthrombosis, and organ transplant rejection comprising the step oftreating a patient with an inhibitor of the PL scramblase activity PLscramblase.

[0095] The present invention is also a method for diagnosing individualswith reduced or elevated capacity for platelet-promoted orerythrocyte-promoted fibrin clot activity by quantitating the level ofcellular expression of PL scramblase in the individual. This method maybe performed by using an antibody to PL scramblase in an immunoblot,ELISA, or fluorescence flow cytometric method. The method may also beperformed using oligonucleotides derived by PL scramblase cDNA in thepolymerase chain reaction. In another embodiment, the method may beperformed by isolating PL scramblase from a whole blood sample,measuring the amount of PL scramblase isolated and comparing themeasurement with a control sample.

[0096] One may wish to use the protein preparation of the presentinvention as a hemostatic agent by topically applying the proteinpreparation to a wound area in a freely bleeding patient.

EXAMPLES

[0097] In the Examples below, we identify the cellular component thatfunctions to mediate the Ca²⁺-dependent reorganization of plasmamembrane phospholipids, we identify the essential structural elements ofthis protein that are required for its phospholipid transportingfunction, and we describe methods for inhibiting or accelerating egressof PS to the surface of activated, injured, or apoptotic cells.

Example 1 Purification, Sequencing and Molecular Cloning of Human PLScramblase

[0098] A. Summary

[0099] The rapid movement of phospholipids (PL) between plasma membraneleaflets is thought to play a key role in expression of plateletprocoagulant activity and in clearance of injured or apoptotic cells.U.S. Ser. No. 08/790,186, upon which this application claims priority,discloses isolation of a ˜37 kDa protein in erythrocyte membrane thatmediates Ca²⁺-dependent movement of PL between membrane leaflets,similar to that observed upon elevation of Ca²⁺ in the cytosol [F.Bassé, et al. J. Biol. Chem. 271:17205-17210, 1996]. Based on internalpeptide sequence obtained from this protein, a 1,445 bp cDNA was clonedfrom a K562 cDNA library. The deduced protein is a proline-rich, type IIplasma membrane protein with a single transmembrane segment near theC-terminus. Antibody against the deduced C-terminal peptide was found toprecipitate the ˜37 kDa red blood cell protein and absorb PL scramblaseactivity, confirming the identity of the cloned cDNA to erythrocyte PLscramblase. Ca²⁺-dependent PL scramblase activity was also demonstratedin recombinant protein expressed from plasmid containing the cDNA.Quantitative immunoblctting revealed an approximately 10-fold higherabundance of PL scramblase in platelet (˜10⁴ molecules per cell) than inerythrocyte (˜10³ molecules/cell), consistent with apparent increased PLscramblase activity of the platelet plasma membrane. PL scramblase mRNAwas found in a variety of hematologic and non-hematologic cells andtissues, suggesting that this protein functions in all cells.

[0100] B. Experimental Procedures

[0101] All experimental procedures and abbreviations are as set out inU.S. Ser. No. 08/790,186, unless otherwise noted. anti-306-318, affinitypurified rabbit antibody (IgG fraction) against peptide [C]ESTGSQEQKSGVW(SEQ ID NO:5); EST, expressed sequence tag; IPTG,isopropyl-β-D-thiogalactopyranoside; MBP, maltose binding protein;

[0102] Materials. Egg yolk phosphatidylcholine (PC), brainphosphatidylserine (PS), 1-palmitoyl 2-oleoyl phosphatidic acid,1-oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-sn-glycero-3-phosphocholine(NBD-PC) and1-oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-sn-glycero-3-phosphoserine(NBD-PS) were obtained from Avanti Polar Lipids. Expressed sequence tag(EST) clone with GenBank accession number gb AA143025 was obtainedthrough American Type Culture Collection (ATCC962235). All restrictionenzymes and amylose resin were from New England BioLabs, Inc. Klentaqpolymerase was from Clontech Laboratories, wheat germ agglutininSepharose from Sigma, IPTG from Eastman Kodak, factor Xa fromHaematologic Technologies, and Bio-Beads SM-2 were from BioRad.N-octyl-p-D-glucopyranoside (OG) and Glu-Gly-Arg chloromethyl ketonewere from Calbiochem. Sodium dithionite (Na₂S₂O₄ Sigma) was freshlydissolved in 1 M Tris pH 10 at a concentration of 1 M.N-Octyl-β-D-glucopyranoside (OG) was purchased from Calbiochem. Sodiumdithionite (Na₂S₂O₄, Sigma) was freshly dissolved in 1 M Tris pH 10 at aconcentration of 1 M.

[0103] PL Scramblase isolation. Human PL scramblase protein and cDNA wasobtained as described in Ser. No. 08/790,186.

[0104] Cloning of PL scramblase into pMAL-C2 expression vector. In orderto express PL scramblase as a fusion protein with maltose bindingprotein (MBP), cDNA encoding PL scramblase was cloned into pMAL-C2 (NewEngland BioLabs). PCR was performed on a full-length clone using theprimers⁵ TCA GAA TTC GGA TCC ATG GAC AAA CAA AAC TCA CAG ATG³ (SEQ IDNO:6) with an EcoR1 site before the ATG start codon and ⁵GCT TGC CTG CAGGTC GAC CTA CCA CAC TCC TGA TTT TTG TTC C³ (SEQ ID NO:7) with a SalIsite after the stop codon. KlenTaq polymerase (Clontech) was used toensure high fidelity amplification. The PCR product was digested withEcoR1 and SalI and isolated by electrophoresis on 1% low melting agarosegel and purification with Wizard kit (Promega). The amplified cDNA wascloned into pMAL-C2 vector digested with EcoRI and SalI, immediately 3′of MBP. This construct was amplified in E. coli strain TB1, and sequenceof the cDNA insert of plasmids from single colonies confirmed.

[0105] Expression and purification of PL scramblase-MBP fusion protein.Ten ml of E. coli TB1 transformed with PL scramblase cDNA-pMAL-C2 wereused to inoculate 1 L of rich LB containing 2 mg/ml glucose, 100 μg/mlampicillin, and the bacteria were allowed to grow for about 4 hours at37° C. When A₆₀₀ reached ˜0.5, IPTG was added to a final concentrationof 0.3 mM. After 2 hours of incubation at 37° C., the cells werecentrifuged at 4000×g for 20 minutes. The cell pellet was suspended in5b ml of 20 mM Tris, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF (columnbuffer), and subjected to a freeze/thaw cycle. After sonication (3×30seconds on ice) and centrifugation at 43,000×g for 1 hour, thesupernatant was applied to 10 ml of amylose resin. The column was washedwith 20 volumes of column buffer, and the scramblase-MBP fusion proteineluted with the same buffer containing 10 mM maltose. Digestion ofMBP-PL scramblase protein with factor Xa was routinely performed at1/100 (w/w) ratio of enzyme and monitored by SDS-PAGE. In addition toMBP, the product of this digest is the PL scramblase translation productcontaining the N-terminal extension Ile-Ser-Glu-Phe-Gly-Phe (codons −6to −1).

[0106] Reconstitution of PL scramblase or scramblase fusion protein intoproteoliposomes. Reconstitution and functional activity were performedessentially as previously described (F. Bassé, et al., J. Biol. Chem.271:17205-17210, 1996; J. G. Stout, et al., J. Clin. Invest.99:2232-2238, 1997). Briefly, a mixture of PC and PS (9:1 molar ratio)was dried under a stream of nitrogen and resuspended in 100 mM Tris, 100mM KCl, 0.1 mM EGTA, pH 7.4 (Tris buffer). Protein samples to bereconstituted were added to the liposomes at a final lipid concentrationof 4 mg/ml in the presence of 60 mM OG, and dialyzed overnight at 4° C.against 200 vol of Tris buffer containing 1 g/L Bio-Beads SM-2. Toliberate PL scramblase from MBP, the proteoliposomes were incubated 3 hat room temperature in the presence of 1/40 (w/w) factor Xa. Thedigestion was terminated by addition of 100 AM Glu-Gly-Arg chloromethylketone. Completeness of the digest was monitored by SDS-PAGE. Followingdialysis, the proteoliposomes were labeled in the outer leaflet byaddition of 0.25 mol % fluorescent NBD-PC (in dimethyl sulfoxide, finalsolvent concentration 0.25%).

[0107] PL Scramblase activity. Scramblase activity was measured aspreviously described (F. Bassé, et al., supra, 1996; J. G. Stout, etal., supra, 1997 and in U.S. Ser. No. 08/790,186). Routinely,proteoliposomes labeled with NBD-PC were incubated for 2 hours at 37° C.in Tris buffer in the presence or absence of 2 mM CaCl₂. Proteoliposomeswere diluted 25-fold in Tris buffer containing 4 mM EGTA and transferredto a stirred fluorescence cuvet at 23° C. Initial fluorescence wasrecorded (SLM Aminco 8000 spectrofluorimeter; excitation at 470 nm,emission at 532 nm), 20 mM dithionite was added, and the fluorescencecontinuously monitored for a total of 120 seconds. The difference innon-quenchable fluorescence observed in presence vs. absence of CaCl₂was attributed to Ca²⁺-induced change in NBD-PC located in the outerleaflet (F. Bassé, et al., supra, 1996; J. G. Stout, et al., supra,1997; J.C. McIntyre and R. G. Sleight, Biochemistry 30:11819-11827,1991). Ionized [Ca²⁺] was calculated using FreeCal version 4.0 software(generously provided by Dr. Lawrence F. Brass, University ofPennsylvania, Philadelphia, Pa.).

[0108] Antibody against PL Scramblase C-terminal peptide. The peptideCESTGSQEQKSGVW (SEQ ID NO:5), corresponding to amino acids 306-318 ofthe predicted open reading frame of PL scramblase with an addedN-terminal cysteine, hemocyanin (Protein Core Facility, Blood ResearchInstitute). Antiserum to this protein was raised in rabbit (CocalicoBiologicals, Inc.) and the IgG fraction isolated on Protein ASepharose-CL4B (Sigma). Peptide-specific antibody was isolated byaffinity chromatography on UltraLink Iodoacetyl beads (Pierce) to whichpeptide CESTGSQEQKSGVW (SEQ ID NO:5) was conjugated. Thisaffinity-purified antibody (anti-306-318) was used forimmunoprecipitation and Western blotting of PL scramblase (below).

[0109] Immunoprecipitation of PL scramblase. PL scramblase purified fromhuman erythrocytes was ¹²⁵I-labeled with Iodogen (Pierce), free iodideremoved by gel filtration, and the protein incubated (4° C., overnight)with either anti-306-318, or an identical quantity of pre-immune rabbitIgG (1 mg/ml in 150 mM NaCl, 10 mM MOPS, 50 mM OG, pH 7.4) or no IgG ascontrol. The IgG was precipitated with protein A Sepharose, washedexhaustively, and protein bands resolved by 8-25% SDS-PAGE (PhastSystem, Pharmacia Biotech Inc.) under reducing conditions. Radioactivebands were visualized by autoradiography. In order to determine whetherantibody to this peptide specifically removed the functional activityassociated with the purified erythrocyte PL scramblase protein, thesupernatant fractions remaining after immunoprecipitation werereconstituted in liposomes for activity measurements, performed asdescribed above. For these experiments, unlabeled erythrocyte PLscramblase substituted for the 125I-labeled protein.

[0110] Western Blot Analysis 2×10⁸ washed platelets, 2×10⁸ erythrocyteghost membranes, 0.9 pmoles of purified recombinant PL scramblase(obtained by factor Xa digest of the PL scramblase-MBP fusion protein),or 0.3 pmoles of PL scramblase purified from human erythrocyte were eachdenatured by boiling in 40 μl sample buffer containing 10% SDS, 4%β-mercaptoethanol, and 1 mM EDTA, and protein bands resolved bySDS-PAGE. After transfer to nitrocellulose, the blocked membrane wasincubated with 1 μg/ml of anti-306-318, and the blot developed withhorseradish preoxidase-conjugated goat anti-rabbit IgG (Sigma) usingChemiluminescence Reagent (Dupont).

[0111] Protein Concentrations. Protein concentrations were estimatedbased upon optical density at 280 nm, using extinction coefficients (M⁻¹cm⁻¹) of 39,000 (PL scramblase), 64,500 (MBP), and 105,000 (PLscramblase-MBP fusion). PL scramblase contained in human platelet anderythrocyte membranes was estimated by quantitative immunoblotting ofthe detergent extracts, with reference to known quantities of purifiedMBP-PL scramblase fusion protein.

[0112] Northern Blot Analysis. Human multiple tissue northern blot andhuman cancer cell line multiple tissue northern blot membranes wereobtained from Clontech. The blots were prehybridized with ExpressHyb(Clontech) at 68° C. for 30 minutes and hybridized with ExpressHybcontaining 5 ng/ml ³²P-labeled PL scramblase cDNA probe at 68° C. for 1hour, then washed and exposed to X-ray film. After development, theblots were stripped and hybridized with ³²P-labeled β-actin cDNA probeusing identical conditions.

[0113] C. Results and Discussion

[0114] U.S. Ser. No. 08/790,186 describes the initial purification of PLscramblase from human erthyrocyte membrane and analysis of cyanogenbromide fragments. The fragments were used to obtain an entire PLscramblase DNA alone (FIG. 1).

[0115]FIG. 1A illustrates the cDNA and deduced amino acid sequence ofhuman PL scramblase. The deduced amino acid sequence of the predictedopen reading frame is shown under the nucleotide sequence. The 32residues of peptide sequence that were obtained from cyanogen bromidedigest of purified erythrocyte PL scramblase are indicated by singleunderline. Also indicated are the residues comprising a predictedinside-to-outside transmembrane domain (Ala291-Gly309; double underline)and protein kinase C phosphorylation site (Thr 161; asterisk). SeeExperimental Procedures for details.

[0116] Analysis of the cDNA-derived protein sequence (Tmpred program,ISREC server, Univ. of Lausanne, Epalinges, Switzerland) revealed astrongly-preferred (p<0.01) inside-to-outside orientation of thepredicted 19 residue transmembrane helix, consistent with a type IIplasma membrane protein. Most of the polypeptide (residues 1-290)thereby extends from the cytoplasmic membrane leaflet, leaving a shortexoplasmic tail (residues 310-318). The predicted orientation of thisprotein is consistent with the anticipated topology of PL scramblase inthe erythrocyte membrane, where lipid-mobilizing function is responsiveto [Ca²⁺] only at the endofacial surface of the membrane (P. Williamson,et al., Biochemistry 31:6355-6360, 1992; E. F. Smeets, et al., Biochim.Biophys. Acta Bio-Membr. 1195:281-286, 1994; F. Bassé, et al., supra,1996; J. G. Stout, et al., supra, 1997; P. Williamson, et al.,Biochemistry 34:10448-10455, 1995; D. L. Bratton, J. Biol. Chem.269:22517-22523, 1994).

[0117] In order to confirm that the cDNA we cloned from the K562 cDNAlibrary actually encodes the same protein purified as PL scramblase fromhuman erythrocyte membrane, we raised a rabbit antibody against thededuced C-terminus predicted from the open reading frame of the clonedcDNA (codons 306-318).

[0118]FIG. 2 illustrates immunoprecipitation of erythrocyte PLscramblase. PL scramblase purified from human erythrocytes wasprecipitated with either anti-306-318 IgG (bar 1), or with pre-immunerabbit IgG (bar 2) and protein remaining in the supernatantreconstituted into liposomes for measurement of residual PL scramblaseactivity. Data normalized to PL scramblase activity measured foridentical controls omitting antibody (100%; bar 3). Error bars denotemean±SD (n=4).

[0119] As shown in FIG. 2, this antibody precipitated the −37 kDa redcell protein we tentatively identified as PL scramblase, and alsoabsorbed the functional activity detected in this isolated erythrocytemembrane protein fraction. We often observed the partial proteolysis of37 kDa PL scramblase to a polypeptide of ˜30 kDa. The apparentsusceptibility of this protein to proteolytic degradation may accountfor the reported rapid loss of activity observed in earlier attempts topurify PL scramblase from platelet (P. Comfurius, et al., Biochemistry35:7631-7634, 1996).

[0120] Expression and membrane reconstitution of recombinant PLscramblase. Recombinant PL scramblase was expressed in E. coli as fusionprotein with MBP, purified by amylose affinity chromatography, andincorporated into PC/PS liposomes for assay of PL scramblase activity.When incorporated into liposomes, the recombinant protein mediated aCa²⁺-dependent transbilayer movement of NBD-PC mimicking the activity ofPL scramblase isolated from erythrocyte. PL scramblase activity wasobserved both for the chimeric MBP-PL scramblase fusion protein, and forrecombinant PL scramblase liberated from MBP through proteolyticdigestion with factor Xa (FIG. 3).

[0121]FIG. 3 depicts an activity assay of recombinant PL scramblase.Purified PL scramblase-MBP fusion protein (0-43×10⁻¹¹ moles; abscissa)was reconstituted into liposomes (1 μmole total PL) and MBPproteolytically removed by incubation with factor Xa in presence of 0.1mM EGTA. After digest to release MBP, the proteoliposomes were recoveredfor determination of PL scramblase activity, measured in the absence (∘)or presence () of 2 mM CaCl₂ as described in Experimental Procedures.Data are corrected for non-specific transbilayer migration of NBD-PCprobe in identically-matched control liposomes containing either MBP orno added protein (<2% NBD-PC sequestered; not shown). Error bars denotemean±SD (n=3). Data of single experiment, representative of two soperformed. Similar results were also obtained for proteoliposomescontaining intact PL scramblase-MBP fusion protein, omitting the factorXa digest (not shown).

[0122] By contrast, no such activity was observed for control proteinconsisting of the pMAL-C2 translation product MBP lacking the PLscramblase cDNA insert. The specific PL mobilizing activity ofrecombinant PL scramblase expressed and purified from E. coli wasapproximately 50% of that observed for the endogenous protein purifiedfrom the erythrocyte membrane, which is likely due to incomplete foldingof the recombinant protein. Half-maximal [Ca²⁺]_(c) required foractivation was approximately 100-200 μM for recombinant protein purifiedfrom E. coli versus −40 μM for the erythrocyte-derived protein, raisingthe possibility that altered folding or an unknown post-translationalmodification in mammalian cells affects the putative Ca2+binding site(F. Bassé, et al., supra, 1996; J. G. Stout, et al., supra, 1997). Inaddition to activation by Ca²⁺, the transbilayer migration of PL inerythrocytes is accelerated upon acidification of the inside leaflet topH<6.0 (in absence of Ca²⁺), a response that is also observed inproteoliposomes containing PL scramblase purified from erythrocytemembranes (J. G. Stout, et al., supra, 1997). A similar acid-dependentactivation of PL mobilizing function was also exhibited byproteoliposomes incorporating recombinant PL scramblase purified from E.coli.

[0123] Platelet PL Scramblase. In addition to the presumed role of PLscramblase in PS exposure following cell injury and upon repeatedsickling of SS hemoglobin red cells, the capacity of activated plateletsto rapidly mobilize aminophospholipids across the plasma membrane isthought to play a central role in the initiation of thrombin generationrequired for plasma clotting (R. F. A. Zwaal and A. J. Schroit, Blood89:1121-1132, 1997). Whereas incubation with Ca²⁺, ionophore causes amarked acceleration in transbilayer movement of plasma membrane PL inboth platelets and erythrocytes, the apparent rate of transbilayer PLmigration in platelet exceeds that in erythrocyte by approximately10-fold, implying either a higher abundance of PL scramblase, or theaction of another component in platelet with enhanced PL scramblingfunction (J. C. Sulpice, et al., J. Biol. Chem. 269:6347-6354; 1994; J.C. Sulpice, et al., Biochemistry 35:13345-13352, 1996). Zwaal andassociates recently reported evidence for the existence of protein(s) inplatelet with functional properties similar to that of PL scramblase weisolated from erythrocyte (F. Bassé, et al., supra, 1996; J. G. Stout,et al., supra, 1997; P. Comfurius, et al., supra, 1996). In order todetermine whether the protein we now identify in the erythrocytemembrane is also found in platelets, we probed platelets with antibodyagainst PL scramblase residues 306-318. FIG. 4 illustratesimmunoblotting of PL scramblase in human erythrocytes and platelets.2×10⁸ platelets (lane 1), and ghost membranes from 2×10⁸ erythrocytes(lane 2), were separated by SDS-PAGE, transferred to nitrocellulose andWestern blotted with anti-306-318 antibody as described in ExperimentalProcedures. Lane 3 contains 0.9 pmoles of factor Xa cleaved recombinantPL scramblase and lane 4 contains 0.3 pmoles of PL scramblase purifiedfrom erythrocytes. Data of single experiment representative of three soperformed.

[0124] As shown in FIG. 4, this antibody blotted a single protein inplatelet with similar mobility to the ˜37 kDa PL scramblase inerythrocyte. Based on quantitative immunoblotting with anti-306-318, weestimate approximately 10⁴ molecules/cell in platelet versus 10³molecules/cell in erythrocyte, consistent with the increased PLscramblase activity and procoagulant function observed for humanplatelets versus erythrocytes.

[0125] Tissue Distribution. In addition to platelet and red blood cell,PL scramblase activity has been observed in many other cells, and thisCa²⁺-induced response is thought to be central to the rapid movement ofPS and phosphatidylethanolamine from inner plasma membrane leaflet tothe surface of perturbed endothelium, and a variety of injured andapoptotic cells (R. F. A. Zwaal and A. J. Schroit, supra, 1997). Theresulting exposure of PS at the cell surface is thought to play a keyrole in removal of such cells by the reticuloendothelial system, inaddition to activation of both the plasma complement and coagulationsystems (R. H. Wang, et al., J. Clin. Invest. 92:1326-1335, 1993; V. A.Fadok, et al., J. Immunol. 148:2207-2216, 1992; R. F. A. Zwaal and A. J.Schroit, supra, 1997). Whereas the molecular mechanism(s) in eachcircumstance remains unresolved, evidence for a specific plateletmembrane protein functioning to accelerate migration of PL betweenmembrane leaflets at increased cytosolic [Ca²⁺] has been reported (P.Comfurius, et al., supra, 1996), similar to the proposed role of PLscramblase in red blood cells (F. Bassé, et al., supra, 1996; J. G.Stout, et al., supra, 1997). It was thus of interest to determinewhether mRNA for this protein is expressed in nucleated cells where PLscramblase-like activity has been observed.

[0126] Northern blotting with PL scramblase cDNA revealed transcripts of˜1.6 and ˜2.6 kb in all tissues and cell lines tested. Sometissue-to-tissue and cell line variability in the relative abundance ofthese two transcripts is apparent, the significance of which remains tobe determined. Also notable was markedly reduced expression in HL-60 andthe lymphoma lines Raji and MOLT-4 whereas abundant message was detectedin spleen, thymus, and peripheral leukocytes. In addition to thetransformed cell lines shown, mRNA for PL scramblase was also confirmedin human umbilical vein endothelial cells. Whereas these data imply thatthe same protein identified as mediating accelerated transbilayerflip-flop of the erythrocyte membrane PL also plays a similar role inthe plasma membrane of platelets, leukocytes and other cells, actualconfirmation for this role of PL scramblase awaits analysis of a cellline that is selectively deficient in this protein. In Scott syndrome, ableeding disorder related to an inherited deficiency of plasma membranePL scramblase function, erythrocytes deficient in PL scramblase activitywere found to contain normal amounts of the PL scramblase protein (J. G.Stout, et al., supra, 1997) and unpublished data). Furthermore, despitethe apparent deficiency in Scott syndrome cells of endogenous PLscramblase function, when PL scramblase protein from these cells waspurified and reconstituted in proteoliposomes containing exogenous PL,it exhibited normal Ca²⁺-dependent PL-mobilizing activity (J. G. Stout,et al., supra, 1997). This suggests that in addition to the knownregulation by intracellular [Ca²⁺], the activity other as yetunidentified membrane or cytoplasmic component (s)

Example 2 Cloning of Murine PL Scramblase and Identity of a ConservedMotif in Phospholipid Scramblase that is Required for AcceleratedTransbilayer Movement of Membrane Phospholipids by Ca²⁺

[0127] A. Summary

[0128] Accelerated transbilayer movement of plasma membranephospholipids (PL) upon elevation of Ca²⁺ in the cytosol plays a centralrole in the initiation of plasma clotting and in phagocytic clearance ofinjured or apoptotic cells. We recently identified a human erythrocytemembrane protein that induces rapid transbilayer movement of PL atelevated Ca²⁺, and presented evidence that this PL scramblase isexpressed in a variety of other cells and tissues where transbilayermovement of plasma membrane PL is promoted by intracellular Ca²⁺ (Q.Zhou, et al., J. Biol. Chem. 272:18240-18244, 1997). We have now clonedmurine PL scramblase for comparison to the human polypeptide (FIG. 1B):Both human and murine PL scramblase are acidic proteins (pI=4.9) with apredicted inside-outside (type 2) transmembrane segment at thecarboxyl-terminus (FIG. 5). Whereas human PL scramblase (318 AA)terminates in a short exoplasmic tail, murine PL scramblase (307 AA)terminates in the predicted membrane-inserted segment. The alignedpolypeptide sequences reveal 65% overall identity, including nearidentity through 12 residues of an apparent Ca²⁺ binding motif(D[A/S]DNFGIQFPLD) spanning residues 273-284 (human, SEQ ID NO:2) and271-282 (murine, SEQ ID NO:4), respectively (FIG. 5). This conservedsequence in the cytoplasmic domain of PL scramblase shows similarity toCa²⁺-binding loop motifs previously identified in known EF-handstructures. Recombinant murine and human PL scramblase were eachexpressed in E. coli and incorporated into proteoliposomes. Measurementof transbilayer movement of NBD-labeled PL confirmed that both proteinscatalyzed Ca²⁺-dependent PL flip/flop similar to that observed for theaction of Ca²⁺ at the cytoplasmic face of plasma membranes. Mutation ofresidues within the putative EF hand loop of human PL scramblaseresulted in loss of its PL mobilizing function, suggesting that theseresidues directly participate in the Ca²⁺ induced active conformation ofthe polypeptide.

[0129] B. Experimental Procedures

[0130] Abbreviations used: PL, phospholipids(s); PC,phosphatidylcholine; PS, phosphatidylserine; NBD-PC,1-oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-sn-glycero-3-phosphocholine;EST, expressed sequence tag; MBP, maltose binding protein; PAGE,polyacrylamide gel electrophoresis; bp, base pair(s); PCR; polymerasechain reaction.

[0131] Materials: Mouse fibroblast 5′-stretch plus cDNA library andKlenTaq polymerase were obtained from CLONTECH Laboratories. Expressedsequence Tag (EST) clone with GenBank™ accession number gb AA110551 wasfrom American Type Culture Collection (ATCC 977052). α-³²P-dCTP waspurchased from Dupont. Random Primed DNA Labeling Kit was fromBoehringer Mannheim. Hybond-N Nylon membrane was from Amersham.Expression vector pMAL-C2, amylose resin and all restriction enzymeswere from New England Biolabs. Wizard Kit was from Promega. QiagenLambda Kit was from Qiagen. Egg yolk phosphatidylcholine (PC), brainphosphatidylserine (PS) and1-oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-sn-glycero-3-phosphocholine(NBD-PC) were obtained from Avanti Polar Lipids. Factor Xa was fromHaematologic Technologies, and Bio-Beads SM-2 were from BioRad.N-octyl-β-D-glucopyranoside and Glu-Gly-Arg chloromethyl ketone werefrom Calbiochem. Sodium dithionite (Sigma) was freshly dissolved in 1MTris, pH 10, at a concentration of 1 M.

[0132] Labeling of DNA Probe: The DNA insert of EST clone gb AA110551was released by digestion with EcoRI and ApalI and purified by WizardKit. Four micrograms of purified DNA were labeled with 1 mCi ofα-³²P-dCTP. The specific radioactivity of the probe was 3.9×10⁸ dpm/μgDNA.

[0133] Isolation of Mouse PL Scramblase cDNA by Plaque Hybridization: E.coli strain Y1090r was transformed by mouse fibroblast cDNA library(6×10⁵ pfu) and poured onto 30 plates (15 cm diameter, 20,000 pfu perplate). Plaques were lifted onto Hybond-N Nylon membranes. Afterdenaturation, neutralization and UV-cross linking, the membranes werefirst prehybridized in a solution composed of 5× Denhardt, 5×SSC, 1%SDS, and 200 μg/ml herring sperm DNA for 3 hours at 68° C., and thenhybridized in the same solution containing 5 ng/ml ³²P-labeled probe for16 hours at 68° C. The membranes were washed once with 2×SSC, 0.1% SDS,then three times with 0.1×SSC, 0.1% SDS for 20 minutes at 65° C., andexposed to X-ray film. Secondary plaque lifts and hybridization werecarried out on 8 positive plaques at a density of about 100plaques/plate. Single positive and well isolated plaques were picked andamplified. λDNA was purified with Qiagen Lambda Maxi Kit.

[0134] DNA Sequencing. DNA was sequenced on an ABI DNA Sequencer Model373 Stretch (Applied Biosystems) using PRISM Ready Reaction DyeDeoxyTerminator Cycle Sequencing Kit (Perkin Elmer).

[0135] Cloning of Mouse PL Scramblase into pMAL-C2 Expression Vector. Inorder to express mouse PL scramblase as a fusion protein with maltosebinding protein (MBP), cDNA encoding mouse PL scramblase was cloned intopMAL-C2 expression vector. PCR was performed on a mouse scramblase cloneusing the primers ⁵TCA GAA TTC GGA TCC ATG GAG GCT CCT CGC TCA GGA AC³(SEQ ID NO:8) with an EcoRI site before the ATG start codon and “GCT TGCCTG CAG GTC GAC CTA CAC ACA GCC TTC AAA AAA CAT G³ (SEQ ID NO:9) with aSalI site after the stop codon. KlenTaq polymerase was used to ensurehigh fidelity amplification. The PCR product was digested with EcoRI andSalI, isolated by electrophoresis, and cloned into pMAL-C2 immediately3′ of MBP. E. coli strain TB1 was transformed, and sequence of the cDNAinsert of plasmid from a single colony was confirmed.

[0136] Expression and Purification of Mouse PL Scramblase-MBP FusionProtein: Mouse PL scramblase was expressed as fusion protein with MBP inE. coli TB1 and purified on amylose resin as previously described forhuman PL scramblase (Q. Zhou, et al., J. Biol. Chem. 272:18240-18244,1997). The purified fusion protein was centrifuged at 106,000×g for 1hour at 4° C. to remove aggregated protein.

[0137] Reconstitution and Functional Activity of PL Scramblase:Reconstitution, removal of MBP, and functional assay of PL scramblasewere performed as previously described (F. Bassé, et al., J. Biol. Chem.271:17205-17210, 1996; Q. Zhou, et al., supra, 1997; J. G. Stout, etal., J. Clin. Invest. 99:2232-2238, 1997). Routinely, 420 pmoles ofprotein were reconstituted with 1 μmol of PL. To remove MBP,proteoliposomes were incubated 3 hours at room temperature with{fraction (1/40)} (w/w) factor Xa. The digest was terminated by additionof 100 μM Glu-Gly-Arg chloromethyl ketone. Proteoliposomes labeled withNBD-PC were incubated for 2 hours at 37° C. in Tris buffer in thepresence or absence of CaCl₂ as indicated in figure legends and diluted25-fold in Tris buffer containing 4 mM EGTA. Initial fluorescence wasrecorded (SLM Aminco 8000 spectrofluorimeter; excitation added, and thefluorescence was continuously monitored for a total of 120 seconds.Scramblase activity was calculated according to the difference innon-quenchable fluorescence observed in presence vs absence of Cacl₂.Ionized [Ca²⁺] was calculated using FreeCal version 4.0 software(generously provided by Dr. Lawrence F. Brass, University ofPennsylvania, Philadelphia, Pa.).

[0138] Protein Concentrations: Protein concentrations were estimatedbased upon optical density at 280 nm, using extinction coefficients (M⁻¹cm⁻¹) of 39,000 (PL scramblase), 64,500 (MBP), and 105,000 (PLscramblase-MBP fusion protein).

[0139] Mutagenesis of PL Scramblase: Human PL scramblase amino acidresidues in EF-hand Ca²⁺-binding motif at positions of Asp²⁷³, Asp²⁷⁵,Phe²⁷⁷ ₁, Ile²⁷⁹, Phe²⁸¹ and Asp²⁸⁴ were mutated to Ala witholigonucleotide-directed mutagenesis by two rounds of PCR. PLscramblase-pMAL-C2 was selected as template, and the first round of PCRwas performed with pairs of a complementary oligonucleotide primercontaining the point mutation plus a primer complementary to a site nearthe ATG initial codon or TAG stop codon. PCR products were purified byWizard kit. Full length mutated PL scramblase cDNA was obtained byoverlapping PCR and cloned back into pMAL-C2 vector. After confirmationof correct DNA sequence the mutants were recombinantly expressed in E.coli as described above and analyzed by SDS-PAGE.

[0140] C. Results and Discussion

[0141] Isolation of cDNA of Mouse PL Scramblase. Murine EST clones inGenBank containing putative PL scramblase sequence were identified by aBlast homology search using the human PL scramblase cDNA. Among severalclones exhibiting significant homology, a 403 bp Stratagene mouse kidneyclone (gb accession number AA110551) with 79% nucleotide sequenceidentity to human PL scramblase was selected and this clone was used toprobe a mouse fibroblast cDNA library. Eight positive clones wereidentified after two rounds of plaque hybridization. Two of the eightclones were sequenced yielding 1354 bp and 1529 bp, respectively.Alignment revealed 1261 bp of overlapping sequence that spanned an openreading frame of 921 bp and specified a total of 1622 bp of unique cDNAsequence (SEQ ID NO:3).

[0142] SEQ ID NO:4 represents the open reading frame of the translatedsequence of SEQ ID NO:3 (see FIG. 1B). The deduced mouse PL scramblasecDNA encodes a 307 residue protein with a molecular weight of 33.9 kDaand a theoretical pI=4.9, similar to values obtained for the humanprotein (318 residues, 35.1 kDa; pI=4.9; ref. (Q. Zhou, et al., supra,1997). The overall identity of the mouse and human PL scramblase is64.8%, with the most divergent sequence generally contained in theN-terminal portion of the polypeptide (FIG. 5). FIG. 5 depicts thealignment of protein sequences of mouse and human PL (HUM) PL scramblasewas performed by FASTA program using the Smith-Waterman algorithm. (W.R.Pearson and D.J. Lipman, Proc. Natl. Acad Sci. USA 85:2444-2448, 1988)Sequence of human PL scramblase is contained in GenBank™ accessionnumber AF008445. Amino acid identities (:) or similarities (.) betweenthe two sequences are indicated. Also indicated are the residuescomprising a predicted inside-out transmembrane domain (MUR 289-307, HUM291-309; double underline), and the 12 residues of the acidic loop of aputative EF-hand (MUR 271-282, HUM v 273-284; single underline).

[0143] In both proteins, a single 19 residue transmembrane helix ispredicted at the carboxyl terminus, exhibiting a strongly preferredinside-to-outside orientation. Whereas the mouse protein terminatesimmediately after this conserved transmembrane helix, the human PLscramblase contains an additional nine residues, implying that the shortexoplasmic peptide in human PL scramblase is non-essential to function.Homology motifs conserved in both proteins include a potential site forprotein kinase C phosphorylation (Thr¹⁵⁹ in mouse, Thr¹⁶¹ in human) anda potential Ca²⁺-binding EF-hand loop motif adjacent to thetransmembrane helix (residue Asp²⁷¹ to Asp¹⁶¹ in mouse and residuesAsp²⁷³ to Asp²⁸⁴ in human). The cytoplasmic orientation of this proteinand the proximity of this putative Ca²⁺-binding domain to the segment ofpolypeptide that is inserted into the plasma membrane are consistentwith the proposed activity of this protein in situ, where Ca²⁺ actingdirectly at the endofacial membrane surface is known to initiate therapid transbilayer movement of plasma membrane PL (P. Williamson, etal., Biochemistry 31:6355-6360, 1992; R. F. A. Zwaal, and A. J. Schroit,Blood 89:1121-1132, 1997; F. Bassé, et al., J. Biol. Chem.271:17205-17210, 1996; D. L. Bratton, J. Biol. Chem. 269:22517-22523,1994; B. Verhoven, et al., Biochim. Biophys. Acta 1104:15-23, 1992).

[0144] Functional Activity of Recombinant Mouse PL Scramblase. In orderto confirm that the cDNA identified as mouse PL scramblase encodes aprotein of similar function to that identified in human, the human andmouse proteins were each expressed in E. coli, purified, andreconstituted in proteoliposomes for measurement of PL mobilizingactivity. Mouse or human PL scramblase-MBP fusion protein (420 pmoles)was reconstituted into PC/PS liposomes (1 μmol total PL), respectively,MBP was removed by digestion of the proteoliposomes with factor Xa, andPL scramblase activity was determined as described under “ExperimentalProcedures” and plotted as a function of external free [Ca²⁺]. Theresults of this experiment indicate that recombinant mouse PL scramblasemediated a Ca²⁺-dependent transbilayer movement of membrane PL with aspecific activity and affinity for Ca²⁺ indistinguishable from thatobserved for the recombinant human protein.

[0145] Mutational Analysis of a Putative Conserved EF-Hand Motif. Asnoted above, the deduced protein sequence of mouse and human PLscramblase reveals an extensive segment of highly conserved sequenceextending through residue Glu³⁰⁶ (in human; corresponding to Glu³⁰⁴ inmouse; FIG. 5). The predicted secondary structure through this portionof the protein reveals that it contains two short alpha-helical segmentsnear the C-terminus that are separated by a 12-residue acidic loop. Inboth proteins (human and mouse), the C-terminal alpha helix represents apredicted transmembrane segment with a strongly-preferredinside-to-outside orientation, whereas sequence contained within theadjacent 12-residue acidic loop conforms in-part to a consensus sequencethat is characteristic of an EF-hand Ca²⁺-binding loop motif (S.Nakayama and R. H. Kretsinger, Annu. Rev. Biophys. Biomol. Struct.23:473-507, 1994). In this motif, residues in positions 1, 3, 5, 7, 9and 12 of the loop contribute to octahedral coordination of the Ca²⁺ion,with the residues in position 1 [Asp], 3[Asp, Asn, or Ser] and 12 [Aspor Glu] being those most highly conserved.

[0146] In order to gain insight into whether this segment of the proteinmight be directly involved in the Ca²⁺-dependent reorganization ofmembrane PL mediated by PL scramblase, we expressed mutant human PLscramblase with Ala substitutions at positions corresponding to residues1 (Asp²⁷³), 3 (Asp²⁷⁵), 5 (Phe²⁷⁷), 7 (Ile²⁷⁹),9 (Phe²⁸¹), and 12(ASp²⁸⁴) of this putative 12 residue EF-hand loop. FIG. 6 illustrates PLscramblase activity as a function of mutational analysis of putative EFhand loop motif contained in human PL scramblase. Wild-type (WT) andmutant constructs of human PL scramblase were expressed as fusionproteins with MBP in E. coli, purified, and reconstituted inproteoliposomes. After release of MBP by incubation with factor Xa, PLscramblase activity was assessed (see “Experimental Procedures”). Foreach mutant construct, the residues in human PL scramblase that werereplaced by Ala are indicated on the abscissa. PL scramblase activity(ordinate) was measured in presence of 2 mM CaCl₂, and in each case wasnormalized to the activity of WT human PL scramblase (11.76±0.44% oftotal NBD-PC flipped), with correction for the non-specific transbilayermovement of NBD-PC (0.20±0.08% of total NBD-PC flipped) measured in PLvesicles lacking added protein. Error bars indicate mean±SD of threeindependent measurements performed with each mutant construct. FIG. 6illustrates the data of single experiment, representative of twoseparate experiments so performed.

[0147] As illustrated by FIG. 6, Ala substitution at any of thesepositions reduced PL scramblase function, with mutation at Asp²⁷⁵resulting in complete inactivation of the Ca²⁺-dependent response. Inthose mutant polypeptides showing partial retention of activity, reducedresponse to Ca²⁺ was related in-part to an apparent reduction in avidityfor Ca²⁺ (FIG. 7). FIG. 7 illustrates the Ca²⁺-dependence of mutanthuman PL scramblase. PL scramblase activity of wild-type (WT) andselected mutant constructs of FIG. 6 was determined as described in“Experimental Procedures” and plotted as a function of external free[Ca²⁺]: WT (); Asp²⁷³ (□); Phe²⁷⁷ (Δ); Ile²⁷⁹ (⋄); Phe²⁸¹(∘) Asp²⁸⁴(∇). The data are corrected for non-specific transbilayer migration ofNBD-PC in the absence of free [Ca²⁺]. Data of single experiment. Theresults described in FIG. 7 suggest that residues contained in theputative EF-hand loop spanning Asp²⁷³-Asp²⁸⁴ are critical to thefunction of PL scramblase, presumably for coordination of Ca²⁺ asrequired to induce the PL transporting state of the protein. It remainsto be determined what conformational changes are induced in thepolypeptide in the presence of Ca²⁺, including potential reorientationof helical segments flanking the putative Ca²⁺ binding loop, that mightcontribute to the accelerated transbilayer movement of membranephospholipids.

Example 3 Plasma Membrane Expression of Phospholipid ScramblaseRegulates Ca²⁺ Induced Movement of Phosphatidylserine to the CellSurface: Alteration of Phosphatidylserine Exposure In Human LymphoblastsThrough Stable Transfection with PL Scramblase cDNA

[0148] A. Summary

[0149] In order to determine whether PL scramblase is responsible forthe rapid movement of PS from inner-to-outer plasma membrane leaflets inother cells exposed to elevated cytosolic [Ca²⁺]_(c), we analyzed howinduced movement of PS to the surface related to cellular content of PLscramblase. Exposure to Ca²⁺ ionophore A23187 resulted in rapid PSexposure in those cells high in PL scramblase (K-562, HEL, 293T, andEBV-transformed lymphocytes), whereas this response was markedlyattenuated in cells with low amounts of the protein (Raji, MOLT-4,HL-60). To confirm this apparent correlation between PL scramblaseexpression and PS egress at elevated [Ca²⁺]_(c), Raji cells weretransfected with PL scramblase cDNA in pEGFP-C2, and stabletransformants expressing various amounts of rGFP-PL scramblase fusionprotein obtained. Clones expressing rGFP-PL scramblase showed plasmamembrane-localized fluorescence and elevated PL scramblase antigenwhereas clones expressing rGFP alone (transfected with pEGFP-C2 withoutinsert) showed only cytoplasmic fluorescence and served as controls. Inabsence of ionophore, expression of rGFP-PL scramblase had no effect oncell viability or background PS exposure. In response to A23187, clonesexpressing GFP-PL scramblase exhibited markedly accelerated movement ofPS to the cell surface when compared to A23187-treated clones expressingGFP with PS movement to the cell surface increasing with amount ofrGFP-PL scramblase expressed. These data indicate that transfection withPL scramblase cDNA promotes [Ca²⁺]_(c)-dependent movement of PS to thecell surface and suggest that this protein normally mediatesredistribution of plasma membrane phospholipids in activated, injured,or apoptotic cells exposed to elevated [Ca²⁺]_(c).

[0150] B. Materials and Methods

[0151] Materials. All restriction enzymes were from New England BioLabs,Inc. (Beverly, Mass.). Klentaq polymerase and pEGFP-C2 vector were fromCLONTECH Laboratories (Palo Alto, Calif.). Bovine coagulation factor Va(FVa), factor Xa (FXa), prothrombin anddansylarginine-N-(3-ethyl-1,5-pentanediyl)amide were from HaematologicTechnologies, Inc. (Essex Junction, Vt.). Chromogenic thrombin substrateS2238 was from DiaPharma Group, Inc. (Franklin, Ohio). Human a-thrombinwas a generous gift from Dr. John W. Fenton (Albany, N.Y.). OPTI-MEM andgeneticin were from Life Technologies (Gaithersburg, Md.). Fetal bovineserum, RPMI 1640, Cell Dissociation Solution, Hank's Balanced SaltSolution (HBSS), Protein A Sepharose-CL4B, leupeptin, and BSA were fromSigma Chemical Co. (St. Louis, Mo.). UltraLink Iodoacetyl resin andSuperSignal ULTRA Chemiluminescence Kit were from Pierce Chemical Co.(Rockford, Ill.). All other chemicals were of reagent grade.

[0152] Cell culture: Human cancer cell lines erythroleukemic HEL,promyelocytic leukemia HL-60, chronic myelogenous leukemia K562,lymphoblastic leukemia MOLT-4, acute T-cell leukemia Jurkat, Burkitt'slymphoma Raji, and megakaryocytic DAMI were from American Type CultureCollection (Rockville, Md.) and cultured in RPMI 1640 containing 10%fetal bovine serum. EBV-transformed cell line W9 established fromperipheral B-lymphocytes of a normal donor was maintained as previouslydescribed (H. Kojima, et al., 1994).

[0153] Antibodies: Anti-GFP: murine monoclonal antibody against greenfluorescent protein (GFP) was from CLONTECH Laboratories. Anti-FVa:murine monoclonal antibody V237 reactive against human or bovine factorVa light chain was the generous gift of Dr. Charles T. Esmon (OklahomaMedical Research Fndn, Oklahoma City, Okla.). Anti-PLScramblase-E306-W318: Rabbit antibody raised against the carboxylterminal peptide sequence E306-W318 of human PL scramblase haspreviously been described (Q. Zhou, et al., supra, 1997). The IgGfraction was isolated on protein A-Sepharose-CL4B and thepeptide-reactive antibody purified by affinity chromatography on peptide[Cys]-ESTGSQEQKSGVW (SEQ ID NO:5) coupled to UltraLink Iodoacetyl resin.

[0154] Plasmid Construction: Human PL scramblase cDNA insert wasreleased from plasmid pMAL-C2-PL scramblase (Q. Zhou, et al., supra,1997) by double cutting with EcoRI and SalI, respectively, and thenligated into pEGFP-C2 vector using the same restriction site. ThepEGFP-C2-PL scramblase plasmid was amplified from single clones in E.coli strain Top 10, and the orientation and reading frame of the insertconfirmed by sequencing on an ABI DNA Sequencer Model 373 Stretch(Perkin Elmer-Applied Biosystems, Foster City, Calif.) using PRISM ReadyReaction DyeDeoxy Terminator Cycle Sequencing Kit.

[0155] Transfection of Raji cells with pEGFP-PL scramblase. 1.6×10⁷ Rajicells were electroporated with 160 μg plasmid DNA (pEGFP-C2-PLscramblase or pEGFP-C2) in a total volume of 0.8 ml OPTI-MEM, using GenePulse Electroporator (Bio-Rad Laboratories, Hercules, Calif.) set at 450V, 500 pF. After 48 hours in culture, 1.5 mg/ml geneticin was added tothe medium and continuously maintained for 4 weeks. Stable transformantsexhibiting GFP fluorescence were sorted by flow cytometry (FACStar,Becton-Dickinson Immunocytometry Systems, San Jose, Calif.) using an FL1sorting gate. The FL1-positive cells were dilutionally cloned in 96 wellculture plates. pEGFP-PL scramblase transformants expressing the 62 kDaGFP-PL scramblase fusion protein were identified by Western blottingwith anti-GFP and with anti-PL-306-W318 antibodies. Western blotting ofpEGFP-C2 transformants (without insert) confirmed presence of 27 kDaGFP. Clones expressing various amounts of GFP-PL scramblase were eachexpanded for functional assay, along with comparable GFP-expressingclones serving as controls.

[0156] Fluorescence Microscopy. Cell clones transfected with pEGPF-C2-PLscramblase or pEGFP-C2 were deposited on glass microscopy slides using aCytospin 3 (Shandon, Inc., Pittsburgh, Pa.). Phase contrast andfluorescence microscopy was performed with a ZEISS AXIOSKOP microscope(Carl Zeiss, Inc., Thornwood, N.Y.) equipped for epifluorescence, andimages were recorded with a MC100 camera system. The exposure times forphotography of fluorescence was 80-200 seconds under automatic controlusing Kodak Ektachrome 1600 film.

[0157] Western Blot Analysis. Western blotting of GFP and PL scramblaseantigens was performed using 1.5×10⁶ cells per lane. After washing inHBSS, supernatants were removed, and the cell pellets extracted with 2%(v/v) NP-40 in 5 mM EDTA, 50 mM benzamidine, 50 mM N-ethyl maleimide, 1mM phenylmethylsulfonyl fluoride, 1 mm leupeptin in HBSS. After removalof insoluble material (250,000×g, 30 minutes, 4° C.), the samples weredenatured at 100° C. in 10% (w/v) SDS sample buffer containing 2%β-mercaptoethanol. Following SDS-PAGE and transfer to nitrocellulose,the blocked membrane was incubated with either 10 μg/ml of rabbitanti-PL scramblase-E306-W318, or 1/10,000 dilution of mouse anti-GFP.The blots were developed with the horseradish peroxidase conjugate ofeither goat anti-rabbit IgG or goat anti-mouse IgG, respectively, usingSuperSignal ULTRA chemiluminescence.

[0158] Measurement of cell surface PS. Calcium ionophore-inducedexposure of PS on the surface of all cell lines analyzed was detected bythe specific binding of coagulation factor Va (light chain) aspreviously described (P. J. Sims, et al., J. Biol. Chem.263:18205-18212, 1988; H. Kojima, et al., J. Clin. Invest. 94:2237-2243,1994). Briefly, cells were washed twice to remove serum proteins andsuspended (2×10⁶ cells/ml) at 37° C. in RPMI 1640 supplemented with 0.1%BSA, 20 mM HEPES, and adjusted to 1.2 mM free [Ca²⁺ ”] At time=0, A23187(0 or 2 μM final concentration) was added from 1 mM stock solution inDMSO, and at times indicated in figure legends, the reaction was stoppedby addition of 6 mM EGTA. PS exposed on the cell surface at each timepoint was detected by incubating (10 minutes, room temperature) 50 μl ofthe cell suspension with 10 μg/ml FVa, followed by 10 μg/ml anti-FVa, todetect the cell-bound FVa light chain. After staining with 10 μg/mlTri-Color conjugated goat anti-mouse IgG (CALTAG Laboratories,Burlingame, Calif.), single-cell fluorescence was quantitated by flowcytometry (FL3 channel, FACScan, Becton Dickinson ImmunocytometrySystems). Use of Tri-Color conjugate to detect cell-bound FVa enabledsimultaneous measurement of cell-associated GFP fluorescence in celllines transformed with the pEGFP-C2 expression plasmid (fluorescence ofGFP detected in FL1 channel). In experiments in which cell lysis wasmonitored by uptake of propidium iodide, cells were stained for boundFVa with FITC-conjugate of goat anti-mouse IgG (FL1 channel)substituting for Tri-Color conjugate, and propidium iodide was detectedin FL3 channel. Propidium iodide (0.5 μg/ml) was added immediatelybefore dilution for flow cytometry.

[0159] Prothrombinase Assay. Prothrombinase activity of Raji cells wasdetermined by modification of methods previously described forplatelets, using the chromogenic thrombin substrate S2238 (P. J. Sims,et al., supra, -1988). Briefly, 1×10⁵ Raji cells (transfected witheither pEGFP-C2 or pEGFP-C2-PL scramblase) were suspended in 200 μl HBSScontaining 1% BSA in the presence of 2 nM FVa, 1.4 μM prothrombin 2.5 mMCaCl₂, and 4 μM dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (toinhibit feed-back activation by thrombin), and incubated at 37° C.Ca²⁺-ionophore A23187 (2 μM), or DMSO (as solvent control) was added,and prothrombin conversion was initiated by addition of 2 nM Fxa.Thrombin generation was stopped after 2 minutes by dilution into 10 mMEGTA and samples were stored on ice. Aliquots were transferred to a96-well plate, and thrombin generated was assayed in TBS containing 1%BSA in presence of 150 AM S2238 by monitoring time-dependent changes inabsorbance at 405 nm using a Thermomax plate reader (Molecular Devices,Sunnyvale, Calif.). Thrombin activity was calculated using purifiedthrombin as standard.

[0160] C. Results

[0161] Analysis of PL scramblase in various human cell lines.Proteoliposomes reconstituted with erythrocyte PL scramblase exhibitaccelerated transbilayer movement of fluorescent phospholipids inresponse to added Ca²⁺, similar to the observed effect of calcium on theendofacial surface of the red cell membrane (Q. Zhou, et al., supra,1997; J. G. Stout, et al., J. Clin. Invest. 99:2232-2238, 1997; Bassé,et al., J. Biol. Chem. 271:17205-17210, 1996). In order to determinewhether this same protein is responsible for mediating the acceleratedegress of plasma membrane PS that is observed under conditions ofelevated cytosolic [Ca²⁺]_(c), we undertook to determine whether thelevel of expression of PL scramblase in various human cell linescorrelated to the induced movement of PS to the surface of these cells.When challenged with a calcium ionophore, human cell lines exhibitconsiderable differences in the extent to which PS is mobilized to thecell surface. Among the cells tested, Raji, HL60, and Dami were notablyunresponsive to A23187, whereas HEL, W9 (an EBV-transformed normalB-lymphocyte), and Jurkat showed notably robust responses. This apparentcell type-specific variability in response to induced elevation of[Ca²⁺]_(c) was consistently maintained through many months of passage inculture, suggesting it reflected an inherent property of each cell line.As shown in FIG. 8, we also observed considerable differences in thecontent of PL scramblase protein among these various cell lines, and thesensitivity of these various cell lines to induced exposure of plasmamembrane PS (lower panel) generally correlated with the amount ofcellular PL scramblase protein detected by Western blotting (upperpanel): Those cell lines that were most responsive to induced elevationof [Ca²⁺]_(c) (HEL, W9, Jurkat) also expressed greatest amounts of PLscramblase antigen, whereas cell lines with attenuated response to[Ca²⁺]_(c) (Raji, HL60, Dami) contained relatively little of thisprotein. Cell lines Molt-4 and K562 showed intermediate responses toelevated [Ca²⁺]_(c) and expressed intermediate levels of PL scramblaseantigen.

[0162]FIG. 8 depicts western blot analysis of PL scramblase in varioushuman cell lines. Constitutive expression of PL scramblase was analyzedin the human cell lines indicated. Upper Panel: Results obtained byWestern blotting with antibody specific for PL scramblase carboxylterminal residues E306-W318 (see Materials & Methods). Each lanecontains the total protein extract of 1.5×10⁶ cells. Lower Panel:Cumulative results of three separate experiments performed as follows:The cells indicated were washed and suspended at 37° C. in the presenceof 1.2 mM free Ca²⁺, and 2 μM A23187 was added. At times shown(abscissa), EGTA was added and cells analyzed for surface exposed PS asdetected by cell-bound FVa light chain (see Materials and Methods). Dataplotted represent the mean increase (±SD) in number of cells thatstained positive for surface PS after 5 minutes incubation withionophore, after correction for initial background of PS-positive cellsbefore addition of ionophore (time=0). Background number of cells thatexposed PS in absence of ionophore was always <15% except in case ofHEL, where this background ranged between 15-30%.

[0163] These relatively large differences in cell line-specificexpression of this protein was also consistently observed despiterepeated passage in culture, and was found to correspond to markeddifferences in level of specific mRNA as detected by Northern blottingwith PL scramblase cDNA (Q. Zhou, et al., supra, 1997), and data notshown). We also noted that those cell lines with the highest content ofPL scramblase generally exhibited a higher background of PS exposed onthe surface in absence of added ionophore. This was most notable for HELfor which approximately 15-30% of the cells were consistently found toexpose PS prior to addition of A23187 (see Discussion).

[0164] Membrane chances underlying ionophore response. In order todetermine whether the increase in PS exposure in ionophore-treated cellsreflected facilitated movement of PS from inner to outer leaflets of theplasma membrane, or, greater sensitivity of the plasma membrane to lyticdisruption, FVa binding to the cell surface was monitored simultaneouslywith uptake of propidium iodide, as a measure of cell lysis. Asillustrated for the human B-lymphocyte lines W9 (high content of PLscramblase) and Raji (low content of PL scramblase), the inducedmovement of PS to the cell surface was found to precede uptake ofpropidium iodide, suggesting that the elevation of [Ca²⁺]_(c) induces acollapse of transmembrane PL asymmetry before onset of lysis. In thecase of Raji cells which are virtually devoid of PL scramblase (see FIG.8), a general insensitivity of the plasma membrane to eitherionophore-induced PS exposure or to lysis was also apparent.

[0165] Transfection of the Raji cell line with pEGFP-C2-PL scramblase.In order to confirm that the extent to which PS moves to the cellsurface with elevation of [Ca²⁺]_(c) actually depends upon the plasmamembrane content of PL scramblase, we stably transformed Raji, a cellline exhibiting low endogenous PL scramblase expression by transfectionwith plasmid pEGFP-C2-PL scramblase. This plasmid expresses PLscramblase as a fusion protein with green fluorescent protein (GFP),facilitating flow cytometric sorting of transformants for subsequentcloning and detection of the expressed recombinant protein in selectedclones. The decision to attach GFP to the amino terminus of PLscramblase was based on prior evidence that the carboxyl terminus of theprotein is membrane inserted and essential for function, and theobservation that other amino terminal fusion constructs of PL scramblaseexpressed in E. coli retained the same activity of the unmodified PLscramblase polypeptide when reconstituted in proteoliposomes (Q. Zhou,et al., supra, 1997), and unpublished data). The expression of thefull-length GFP-PL scramblase fusion protein in selected transformedclones was confirmed by Western blotting with antibody specific for GFP,and with antibody raised against peptide sequence of the carboxylterminus of human PL scramblase. As illustrated by fluorescencemicrographs shown in FIG. 9, clones that expressed the GFP-PL scramblasefusion protein showed a distinct rim appearing pattern of fluorescence,consistent with trafficking of GFP-PL scramblase to the plasma membrane.FIG. 9 illustrates fluorescence micrographs of GFP-PL scramblasetransformed Raji cells. Fluorescence photomicrography of GFPfluorescence expressed in the transformed Raji clones was performed asdescribed in Materials and Methods. FIG. 9A shows fluorescence of cellsexpressing GFP; FIG. 9B shows cells transfected with pEGFP-C2-PLscramblase plasmid and expressing GFP-PL scramblase fusion protein. Dataof single experiment, representative of results obtained for all clonestransfected with either pEGFP-C2 or pEGFP-C2-PL scramblase. By contrast,clones that expressed GFP alone exhibited diffuse fluorescencethroughout the cytoplasm, with no obvious staining of the plasmamembrane. These data provide the first direct evidence that PLscramblase cDNA encodes a protein that predominantly trafficks to theplasma membrane under normal conditions of cell growth.

[0166] Analysis of PS mobilizing function in GFP-PL scramblasetransformants. After geneticin selection, clonal populations oftransformed Raji cells expressing comparable levels of either GFP-PLscramblase or GFP (transformed with pEGFP-C2 lacking insert) wereanalyzed for their capacity to mobilize PS to the cell surface. Inresponse to an A23187-induced elevation of [Ca²⁺]_(c), transformantsexpressing the GFP-PL scramblase fusion construct showed a markedincrease in both the rate and extent that PS became exposed on the cellsurface, when compared to either the identically-treated parental Rajicell line or to GFP-expressing clones transformed with pEGFP-C2 vectoralone. As was also evident from these data, in the absence of ionophore,we consistently noted a small but reproducible increase in thebackground level of PS exposure in transformants expressing GFP-PLscramblase protein, when compared to either the parental Raji cell linesor to GFP-expressing clones transformed with vector alone.

[0167] Induction of membrane procoagulant function through expression ofPL scramblase. In order to confirm that the increased expression of FVabinding sites detected upon activation of GFP-PL scramblase transformedclones reflected an increase in the procoagulant (clot-promoting)properties of the plasma membrane of these cells, the capacity of GFP-PLscramblase transformed cells to provide catalytic membrane surface forthe prothrombinase (FVaXa) enzyme complex was compared to clonesexpressing GFP alone. These data confirmed that expression ofrecombinant PL scramblase in the Raji cell line was also accompanied byan increase in cell capacity to catalyze the prothrombinase reactionupon entry of calcium into the cytosol.

[0168] Level of expression of PL scramblase regulates capacity of tomobilize PS to the cell surface. In order to confirm the apparentcorrelation between endogenous cell content of PL scramblase and plasmamembrane sensitivity to elevated [Ca²⁺]_(c) that is evident whendifferent human cell lines are compared (see FIG. 8), we analyzedmultiple Raji clones that were stably transfected with either GFP-PLscramblase or with GFP vector alone (FIG. 10). FIG. 10 illustrates thatthe level of expression of PL scramblase determines plasma membranesensitivity to intracellular Ca²⁺. The relationship between level ofrecombinant protein expressed (GFP fluorescence detected in FL1 channel;abscissa) and numbers of cells that expose PS after 2 minutes incubationwith A23187 (ordinate) is plotted for multiple transformed Raji clones.Analysis was gated to include only those cells distinctly positive forGFP fluorescence (FL1 channel), and cell-bound FVa was stained withTri-color conjugate and detected in FL3 channel (see Materials andMethods). Analysis was performed on all cells positive for GFPfluorescence. Open symbols indicate individual clones stably transformedby transfection with pEGFP-C2; closed symbols indicate individual clonesstably transformed with pEGFP-PL scramblase. Data of single experiment,representative of three so performed. These experiments confirm that thecapacity of GFP-PL scramblase transformants to mobilize PS to the cellsurface generally correlates with the amount of the expressed GFP-PLscramblase fusion protein, whereas this cell response to increased[Ca²⁺]_(c) is unaffected by cell content of GFP. In addition toconfirming the role of PL scramblase in the plasma membrane response to[Ca²⁺]_(c), these data suggest that the capacity to mobilize PS to thecell surface and thereby support plasma clotting in activated, injuredor apoptotic cells exposed to elevated [Ca²⁺]_(c) can be altered bychanging the level of expression of PL scramblase expressed in theplasma membrane.

[0169] Discussion

[0170] These results provide the first evidence that the PL scramblaseprotein identified in the erythrocyte membrane and implicated in[Ca²⁺]_(c)-induced remodeling of membrane phospholipids actuallyfunctions to induce accelerated transbilayer movement of plasma membranephospholipid in human cells that express this protein. Our results alsoconfirm that the level of expression of plasma membrane PL scramblasecan determine the extent to which PS is mobilized to the cell surfaceupon elevation of [Ca²⁺]_(c), and suggest that this protein normallyfunctions to mediate the redistribution of plasma membrane phospholipidsin response to the entry of calcium into the cytosol. Furthermore, thesedata provide the first indication that the movement of PS and otherprocoagulant aminophospolipids from plasma membrane inner leaflet to thecell surface can be manipulated by selectively altering the level ofexpression of a particular cellular protein, either through directtransfection with the PL scramblase cDNA, or potentially, by anotherintervention affecting cellular expression of functional PL scramblase

[0171] Whereas these experiments suggest that direct activation ofplasma membrane PL scramblase is responsible for the increased cellsurface exposure of PS that is observed in various activated, injured orapoptotic cells exposed to elevated [Ca²⁺]_(c), we cannot exclude thepossibility.that there are other cellular components that contribute tothe accelerated movement of PS from inner to outer plasma membraneleaflet under these conditions. In particular, whereas PL scramblase hasbeen shown to mediate the bidirectional movement of PS and otherphospholipids between membrane leaflets, it has been suggested thatthere is also a PS-selective pathway in the platelet plasma membrane,designated “PS floppase”, which mediates vectorial movement of PS frominner to outer plasma membrane leaflet (P. Gaffet, et al., Biochemistry34:6762-6769, 1995). Experimental evidence for the existence of thisvectorial and headgroup-selective PS floppase pathway in platelet orother cell membranes remains controversial (R. F. A. Zwaal, et al.,supra, 1997; P. Williamson, et al., Biochemistry 31:6355-6360, 1995;C.-P. Chang, et al., J. Biol. Chem. 268:7171-7178, 1993), and awaitsidentity of a [Ca²⁺]_(c)-activated and PS-selective transporter that isdistinct from the plasma membrane PL scramblase found in platelets anderythrocytes, a protein that does not exhibit apparent selectivity forthe PS headgroup (J. G. Stout, et al., supra, 1997; P. Comfurius, etal., Biochemistry 35, 7631-7634, 1996; F. Bassé, et al., supra, 1996).

[0172] In addition to conferring increased sensitivity of the plasmamembrane to ionophore-induced elevation of [Ca²⁺]_(c), we generallyobserved a higher background of PS exposure (in absence of ionophore) inthose transfected cell clones expressing large amounts of the GFP-PLscramblase fusion protein. This elevated background PS exposure was alsoobserved in the case of untreated HEL, the cell line containing thehighest endogenous content of PL scramblase. Although we suspect thatthis increased background reflects the enhanced sensitivity of theplasma membrane of these cells to any adventitial elevation of[Ca²⁺]_(c) during cell processing for assay, we cannot exclude thepossibility that these cells are also inherently more fragile due to thelarge amounts of PL scramblase that is inserted into the plasmamembrane.

[0173] While the movement of plasma membrane PS to the cell surface atelevated [Ca²⁺]_(c) can be demonstrated in a variety of cells andtissues (R. F. A. Zwaal, et al., supra, 1997; P. Devaux, supra, 1991),we detect marked differences in the levels of PL scramblase mRNA andprotein among different human cell types, which is generally reflectedby corresponding differences in sensitivity to this [Ca²⁺]_(c)-inducedcollapse of plasma membrane PL asymmetry (see FIG. 8, and Q. Zhou, etal., supra, 1997). Although the transcriptional regulation of the PLscramblase gene remains to be determined, it is of interest to note thatsuch cell or tissue-specific differences in PL scramblase expression hasthe potential to significantly affect the biological properties of thecell. In particular, we note that the content of PL scramblase in humanplatelet is approximately 10-fold greater than that of the erythrocyte,which is consistent with the respective PS-mobilizing potential anddifferent roles of these two cells in contributing procoagulant membranesurface for thrombin generation during blood clotting (Q. Zhou, et al.,supra, 1997). In addition to the relatively high levels of PL scramblaseidentified in circulating human platelets, this protein was also mostabundant in the cell line HEL, whereas only small amounts of thisprotein (and low PL scramblase activity) was detected for Dami (FIG. 8),two human cancer cell lines exhibiting partial megakaryocytic-likeproperties. It is also noteworthy that several of the lymphoma-derivedcell lines (e.g., Raji, MOLT-4) express considerably reduced levels ofPL scramblase, and also show distinctly attenuated PS exposure inresponse to elevated [Ca²⁺]_(c), when compared to either peripheralblood leukocytes or to EBV-transforms of normal lymphocytes (FIG. 8).The collapse of plasma membrane phospholipid asymmetry is a relativelyearly event in apoptosis of lymphocytes and other cells, and theconsequent exposure of PS on the cell surface is thought to contributeto phagocytic removal of such cells by scavenger macrophages (V. A.Fadok, et al., J. Immunol. 148:2207-2216, 1992; B. Verhoven, et al., J.Exp. Med. 182:1597-1601, 1995). It is therefore of interest to considerwhether the apparent resistance of certain lymphoma-derived cell linesto such [Ca²⁺]_(c)-induced remodeling of plasma membrane phospholipidsmight contribute to the proliferative potential and in vivo survival ofthese or other transformed cells.

Example 4 Inactivation of Human PL Scramblase by Treatment With theThiolester Cleaving Reagent, Hydroxylamine

[0174] A. Summary

[0175] Incubation of human erythrocyte PL scramblase with hydroxylamineunder conditions known to favor hydrolysis of protein cysteinyl-fattyacyl bonds was found to cause near complete loss of PL scramblase'sfunction in promoting movement of PL between membrane leaflets. Thesedata suggest that for normal activity, the PL scramblase polypeptiderequires post translational modification through addition of athiolester-linked fatty acid. Furthermore, these data imply that methodsthat either prevent cellular acylation of the polypeptide, or thatcleave cysteinyl thiolester linkages, will effectively inhibitendogenous PL scramblase activity.

[0176] B. Methods

[0177] Protein purification. PL scramblase was purified from humanerythrocyte ghost membranes as previously described (F. Bassé, et al.,supra, 1996; J. G. Stout, et al., supra, 1997).

[0178] Treatment with Hydroxylamine. PL scramblase was incubated 1 hourroom temperature in 1 M hydroxylamine, -25 mM octylglucoside, 1 MTris-HCl at pH 7.4. Match control samples of the protein wereidentically incubated under these conditions, omitting hydroxylamine.After incubation, samples were dialyzed and reconstituted into PLproteoliposomes for assay of PL scramblase activity.

[0179] Membrane reconstitution and assay. PL scramblase wasreconstituted into proteliposomes and activity determined as previouslydescribed(F. Bassé, et al., supra, 1996; J. G. Stout, et al, supra,1997).

[0180] C. Results and Discussion

[0181] As shown in FIG. 11, incubation with hydroxylamine resulted innearly complete inactivation of PL scramblase. FIG. 11 illustratesinactivation of PL scramblase by hydroxylamine. Purified humanerythrocyte PL scramblase was incubated 1 hour, room temperature, in thepresence of 50 mM octylglucoside, 1M TrisHCl, and either 0 (control) or1M (hydroxylamine) hydroxylamine at pH 7.2. Untreated refers to samplemaintained in low ionic strength sample buffer at 4° C. Each sample wasthen dialyzed and reconstituted into proteoliposomes for assay of PLscramblase activity using NBD-PC as detailed in Bassé, et al., supra,1996 with modifications of Stout, et al., supra, 1997. Ordinateindicates percent of total NBD-PC flipped during 3 hours. Incubation wasin 2 mM Ca ²⁺, with correction for background measured in 0.1 mM EGTA.The error bars denote mean±SD, n=7. Combined data of three independentexperiments performed on different days.

[0182] Because the conditions of incubation (neutral pH) were chosen tofavor specific cleavage of cysteinyl thioester bonds without disulfidebond reduction, these results imply an essential thioester linkagewithin the protein. In a membrane-associated protein with cytoplasmicCys residues, such as found in erythrocyte PL scramblase, thisthiolester bond is normally provided by palmitic acid in ester linkageto one or more cysteinyl thiols.(H. Schroeder, et al., J. Cell Biol.134:647-660, 1996; M. Stauffenbiel, J. Biol. Chem. 263:13615-13622,1988; C. A. Wilcox, et al., Biochemistry 26:1029-1036, 1987). Whereasthe possibility of disulfide reduction by hydroxylamine cannot beexcluded, it is important to note that (1) virtually all cysteineresidues in PL scramblase are normally exposed to cytoplasmic reducingagents such as glutathione, and disulfide bonds formation is thereforenot anticipated and (2) The absence of any functionally-importantdisulfide bonds in PL scramblase can be assumed based on the retentionof normal PL scramblase activity when the protein was incubated invarious reducing agents, including dithiothreitol (F. Bassé, et al.,supra, 1996; J. G. Stout, et al., supra, 1997). Thus these data suggestthat PL scramblase polypeptide requires post-translational modificationthrough addition of a thiolester-linked fatty acid for its normalfunction in the plasma membrane. Furthermore, these data imply thatreagents that either prevent cellular acylation of the polypeptide, or,reagents that cleave cysteinyl thiolester linkages, will effectivelyinhibit endogenous PL scramblase activity.

1 9 1 1445 DNA Homo sapiens 1 cgcggccgcg tcgaccgaaa ccaggagccgcgggtgttgg cgcaaaggtt actcccagac 60 ccttttccgg ctgacttctg agaaggttgcgcagcagctg tgcccgacag tctagaggcg 120 cagaagagga agccatcgcc tggccccggctctctggacc ttgtctcgct cgggagcgga 180 aacagcggca gccagagaac tgttttaatcatggacaaac aaaactcaca gatgaatgct 240 tctcacccgg aaacaaactt gccagttgggtatcctcctc agtatccacc gacagcattc 300 caaggacctc caggatatag tggctaccctgggccccagg tcagctaccc acccccacca 360 gccggccatt caggtcctgg cccagctggctttcctgtcc caaatcagcc agtgtataat 420 cagccagtat ataatcagcc agttggagctgcaggggtac catggatgcc agcgccacag 480 cctccattaa actgtccacc tggattagaatatttaagtc agatagatca gatactgatt 540 catcagcaaa ttgaacttct ggaagttttaacaggttttg aaactaataa caaatatgaa 600 attaagaaca gctttggaca gagggtttactttgcagcgg aagatactga ttgctgtacc 660 cgaaattgct gtgggccatc tagaccttttaccttgagga ttattgataa tatgggtcaa 720 gaagtcataa ctctggagag accactaagatgtagcagct gttgttgtcc ctgctgcctt 780 caggagatag aaatccaagc tcctcctggtgtaccaatag gttatgttat tcagacttgg 840 cacccatgtc taccaaagtt tacaattcaaaatgagaaaa gagaggatgt actaaaaata 900 agtggtccat gtgttgtgtg cagctgttgtggagatgttg attttgagat taaatctctt 960 gatgaacagt gtgtggttgg caaaatttccaagcactgga ctggaatttt gagagaggca 1020 tttacagacg ctgataactt tggaatccagttccctttag accttgatgt taaaatgaaa 1080 gctgtaatga ttggtgcctg tttcctcattgacttcatgt tttttgaaag cactggcagc 1140 caggaacaaa aatcaggagt gtggtagtggattagtgaaa gtctcctcag gaaatctgaa 1200 gtctgtatat tgattgagac tatctaaactcatacctgta tgaattaagc tgtaaggcct 1260 gtagctctgg ttgtatactt ttgcttttcaaattatagtt tatcttctgt ataactgatt 1320 tataaaggtt tttgtacatt ttttaatactcattgtcaat ttgagaaaaa ggacatatga 1380 gtttttgcat ttattaatga aacttcctttgaaaaactgc tttaaaaaaa agtcgacgcg 1440 gccgc 1445 2 318 PRT Homo sapiens2 Met Asp Lys Gln Asn Ser Gln Met Asn Ala Ser His Pro Glu Thr Asn 1 5 1015 Leu Pro Val Gly Tyr Pro Pro Gln Tyr Pro Pro Thr Ala Phe Gln Gly 20 2530 Pro Pro Gly Tyr Ser Gly Tyr Pro Gly Pro Gln Val Ser Tyr Pro Pro 35 4045 Pro Pro Ala Gly His Ser Gly Pro Gly Pro Ala Gly Phe Pro Val Pro 50 5560 Asn Gln Pro Val Tyr Asn Gln Pro Val Tyr Asn Gln Pro Val Gly Ala 65 7075 80 Ala Gly Val Pro Trp Met Pro Ala Pro Gln Pro Pro Leu Asn Cys Pro 8590 95 Pro Gly Leu Glu Tyr Leu Ser Gln Ile Asp Gln Ile Leu Ile His Gln100 105 110 Gln Ile Glu Leu Leu Glu Val Leu Thr Gly Phe Glu Thr Asn AsnLys 115 120 125 Tyr Glu Ile Lys Asn Ser Phe Gly Gln Arg Val Tyr Phe AlaAla Glu 130 135 140 Asp Thr Asp Cys Cys Thr Arg Asn Cys Cys Gly Pro SerArg Pro Phe 145 150 155 160 Thr Leu Arg Ile Ile Asp Asn Met Gly Gln GluVal Ile Thr Leu Glu 165 170 175 Arg Pro Leu Arg Cys Ser Ser Cys Cys CysPro Cys Cys Leu Gln Glu 180 185 190 Ile Glu Ile Gln Ala Pro Pro Gly ValPro Ile Gly Tyr Val Ile Gln 195 200 205 Thr Trp His Pro Cys Leu Pro LysPhe Thr Ile Gln Asn Glu Lys Arg 210 215 220 Glu Asp Val Leu Lys Ile SerGly Pro Cys Val Val Cys Ser Cys Cys 225 230 235 240 Gly Asp Val Asp PheGlu Ile Lys Ser Leu Asp Glu Gln Cys Val Val 245 250 255 Gly Lys Ile SerLys His Trp Thr Gly Ile Leu Arg Glu Ala Phe Thr 260 265 270 Asp Ala AspAsn Phe Gly Ile Gln Phe Pro Leu Asp Leu Asp Val Lys 275 280 285 Met LysAla Val Met Ile Gly Ala Cys Phe Leu Ile Asp Phe Met Phe 290 295 300 PheGlu Ser Thr Gly Ser Gln Glu Gln Lys Ser Gly Val Trp 305 310 315 3 1622DNA Mus musculus 3 tctaaagact caggaaacaa aacctaaatt gcctcaaagttcaggtgctt tttctccctg 60 actttagtct agtggagtag tgcagcacct atgcctttctgagaggagtc tggagagctg 120 agtcgctgct ggtgctagga ttctaggaat tcgcctcacttggagctgca tgagaaaaga 180 aaggcttgca aatggaggct cctcgctcag gaacatacttgccagctggg tatgcccctc 240 agtatcctcc agcagcagtc caaggacctc cagagcatactggacgcccc acattccaga 300 ctaactacca agttccccag tctggttatc caggacctcaggctagctac acagtctcaa 360 catctggaca tgaaggttat gctgctacac ggcttcctattcaaaataat cagactatag 420 tccttgcaaa cactcagtgg atgccagcac caccacctattctgaactgc ccacctgggc 480 tagaatactt aaatcagata gatcagcttc tgattcatcagcaagttgaa cttctagaag 540 tcttaacagg ctttgaaaca aataacaaat ttgaaatcaagaacagcctc gggcagatgg 600 tttatgttgc agtggaagat actgactgct gtactcgaaattgctgtgaa gcgtctagac 660 ctttcacctt aagaatcctg gatcatctgg gccaagaagtcatgactctg gagcgacctc 720 tgagatgcag tagctgctgc ttcccctgct gcctccaggagatagaaatc caggctcctc 780 cgggggtgcc aataggttat gtgactcaga cctggcacccatgtctgcca aagctcactc 840 ttcagaacga caagagggag aatgttctaa aagtagttggtccatgtgtt gcatgcacct 900 gctgttcaga tattgacttt gagatcaagt ctcttgatgaagtgactaga attggtaaga 960 tcaccaagca gtggtctggt tgtgtgaaag aggccttcacggattcggat aactttggga 1020 tccaattccc gctagacctg gaggtgaaga tgaaagctgtgacgcttggt gcttgcttcc 1080 tcatagatta catgtttttt gaaggctgtg agtaggaacagaaatccgac ctgcagtagg 1140 aatcaatgaa agaggacaga gaagatctga agtctacacaaggagatcat atgattgaga 1200 gacctggggc tttttgattt cttcattgaa atttctcagaatcaagctgt tatacatgaa 1260 gcatagtatg taacattttg gttttcaaat ggtagtttatcttttacatt attggaatag 1320 acctggataa ttatctttat acacttctaa aaatatgcaccaaattcaag ttaaaaaaaa 1380 aaagacgaag agaagtgtat gttttaaaat aaaacattttatggaaaagt aagttaaatc 1440 ataatctggg atttattttt catcttttgt tcaatttaaaccttgttagt gctgatttta 1500 ttataaaatt gtactttact atcaaaccta gttagtttatttcttacaga aatcctccta 1560 ttattttgaa attacatatt tttgaaagct ttttaaaagatactattgcc tgggaaattc 1620 ta 1622 4 307 PRT Mus musculus 4 Met Glu AlaPro Arg Ser Gly Thr Tyr Leu Pro Ala Gly Tyr Ala Pro 1 5 10 15 Gln TyrPro Pro Ala Ala Val Gln Gly Pro Pro Glu His Thr Gly Arg 20 25 30 Pro ThrPhe Gln Thr Asn Tyr Gln Val Pro Gln Ser Gly Tyr Pro Gly 35 40 45 Pro GlnAla Ser Tyr Thr Val Ser Thr Ser Gly His Glu Gly Tyr Ala 50 55 60 Ala ThrArg Leu Pro Ile Gln Asn Asn Gln Thr Ile Val Leu Ala Asn 65 70 75 80 ThrGln Trp Met Pro Ala Pro Pro Pro Ile Leu Asn Cys Pro Pro Gly 85 90 95 LeuGlu Tyr Leu Asn Gln Ile Asp Gln Leu Leu Ile His Gln Gln Val 100 105 110Glu Leu Leu Glu Val Leu Thr Gly Phe Glu Thr Asn Asn Lys Phe Glu 115 120125 Ile Lys Asn Ser Leu Gly Gln Met Val Tyr Val Ala Val Glu Asp Thr 130135 140 Asp Cys Cys Thr Arg Asn Cys Cys Glu Ala Ser Arg Pro Phe Thr Leu145 150 155 160 Arg Ile Leu Asp His Leu Gly Asn Glu Val Met Thr Leu GluArg Pro 165 170 175 Leu Arg Cys Ser Ser Cys Cys Phe Pro Cys Cys Leu GlnGlu Ile Glu 180 185 190 Ile Gln Ala Pro Pro Gly Val Pro Ile Gly Tyr ValThr Gln Thr Trp 195 200 205 His Pro Cys Leu Pro Lys Leu Thr Leu Gln AsnAsp Leu Arg Glu Asn 210 215 220 Val Leu Lys Val Val Gly Pro Cys Val AlaCys Thr Cys Cys Ser Asp 225 230 235 240 Ile Ser Phe Glu Ile Lys Ser LeuAsp Glu Val Thr Arg Ile Gly Leu 245 250 255 Ile Thr Leu Gln Trp Ser GlyCys Val Leu Glu Ala Phe Thr Asp Ser 260 265 270 Asp Asn Phe Gly Ile GlnPhe Pro Leu Asp Leu Glu Val Lys Met Lys 275 280 285 Ala Val Thr Leu GlyAla Cys Phe Leu Ile Asp Tyr Met Phe Phe Glu 290 295 300 Gly Cys Glu 3055 14 PRT Artificial Sequence Description of Artificial Sequence Peptideused to affinity purify 306-318 antibodies 5 Cys Glu Ser Thr Gly Ser GlnGlu Gln Lys Ser Gly Val Trp 1 5 10 6 39 DNA Artificial SequenceDescription of Artificial Sequence PL scramblase primer 6 tcagaattcggatccatgga caaacaaaac tcacagatg 39 7 43 DNA Artificial SequenceDescription of Artificial Sequence PL scramblase primer 7 gcttgcctgcaggtcgacct accacactcc tgatttttgt tcc 43 8 38 DNA Artificial SequenceDescription of Artificial Sequence PL scramblase primer 8 tcagaattcggatccatgga ggctcctcgc tcaggaac 38 9 43 DNA Artificial SequenceDescription of Artificial Sequence PL scramblase primer 9 gcttgcctgcaggtcgacct acacacagcc ttcaaaaaac atg 43

We claim:
 1. A preparation of phospholipid scramblase, wherein theprotein is approximately 35 kD as measured on a 12.5% SDS-polyacrylamidegel under reducing conditions.
 2. The preparation of claim 1 wherein thescramblase is human phospholipid scramblase.
 3. The preparation of claim1 wherein the protein comprises SEQ ID NO:2.
 4. The preparation of claim1 wherein the protein comprises amino acid residues 85-309 of SEQ IDNO:2
 5. The preparation of claim 1 wherein the scramblase is mousephospholipid scramblase.
 6. The preparation of claim 1, wherein thephospholipid scramblase comprises SEQ ID NO:4.
 7. The preparation ofclaim 1, wherein the phospholipid scramblase comprises amino acidresidues 83-307 of SEQ ID NO:4
 8. The preparation of claim 5 wherein theprotein is isolated from mouse erythrocyte membranes.
 9. The preparationof claim 1 wherein the protein is produced by cells genetically modifiedso as to express the protein.
 10. The preparation of claim 9 wherein theprotein is produced by an organism selected from the group consisting ofbacteria, insect cells, and yeast.
 11. A recombinant DNA sequenceencoding PL scramblase.
 12. The sequence of claim 11, wherein thesequence is SEQ ID NO:1.
 13. The sequence of claim 11, comprisingnucleotides 211-1164 of SEQ ID NO:1.
 14. The sequence of claim 11,comprising nucleotides 463-1137 of sequence ID NO:1.
 15. The sequence ofclaim 11, wherein the sequence is SEQ ID NO:3.
 16. The sequence of claim11, comprising nucleotides 192-1112 of SEQ ID NO:3.
 17. The sequence ofclaim 11, comprising nucleotides 438-1112 of SEQ ID NO:3.
 18. Thesequence of claim 11, wherein the sequence is SEQ ID NO:1 and whereinthe sequence is part of a protein expression vector.
 19. The sequence ofclaim 11, comprising nucleotides 211-1164 of SEQ ID NO:1 and wherein thesequence is part of a protein expression vector.
 20. The sequence ofclaim 11, comprising nucleotides 463-1137 of SEQ ID NO:1 and wherein thesequence is part of a protein expression vector.
 21. The sequence ofclaim 11, wherein the sequence is SEQ ID NO:3 and wherein the sequenceis part of a protein expression vector.
 22. The sequence of claim 11,wherein the sequence comprises 192-1112 of SEQ ID NO:3 and wherein thesequence is part of a protein expression vector.
 23. The sequence ofclaim 11, comprising nucleotides 438-1112 of SEQ ID NO:3 and wherein thesequence is part of a protein expression vector.
 24. The sequence ofclaim 11 wherein the sequence has been modified to preventpost-translational.
 25. The sequence of claim 24, wherein the mutantphospholipid scramblase comprises a non-functionally equivalentsubstitution of one or more residues that are phosphorylated by cellularprotein kinases, and said residues are selected from the groupconsisting of Threonine, Serine, or Tyrosine.
 26. The sequence of claim24 wherein the sequence comprises a mutation with a non-functionalequivalent substitution of residue Thr161 of SEQ ID NO:2; Thr159 of SEQID NO:4 or the equivalent residue in the conserved region of another PLscramblase.
 27. The sequence of claim 24 wherein the sequence comprisesat least one non-functional equivalent substitution within residuesAsp273-Asp284 of SEQ ID NO:2; Asp271-Asp282 of SEQ ID NO:4 or theequivalent residue in the conserved region of another PL scramblase. 28.The sequence of claim 24 wherein the sequence comprises a mutation atCys297 of SEQ ID NO:2 or the equivalent residue in the conserved regionof another PL scramblase.
 29. A protein encoded by the sequence of claim11.
 30. A protein encoded by the sequence of claim
 24. 31. A proteinencoded by the sequence of claim
 25. 32. A protein encoded by thesequence of claim
 26. 33. A protein encoded by the sequence of claim 27.34. A protein encoded by the sequence of claim
 28. 35. An animalgenetically engineered to eliminate expression of phospholipidscramblase in all germ line cells.
 36. The animal of claim 35 whereinthe animal is selected from the group consisting of mouse and pig.
 37. Amethod of inhibiting expression of the coagulant properties of theplasma membrane of a cell comprising the step of expressing in the cellplasma membrane a mutant PL scramblase, wherein the PL scramblase has areduced activity in mediating transmembrane movement of plasma membranephospholipids.
 38. The method of claim 37 wherein the cell is a part ofa tissue or organ.
 39. The method of claim 37, wherein the mutantphospholipid scramblase comprises a non-functionally equivalentsubstitution of one or more residues that are normally phosphorylated bycellular protein kinases, and said residues are selected from the groupThreonine, Serine, or Tyrosine.
 40. The method of claim 37, wherein themutant phospholipid scramblase comprises a non-functionally equivalentsubstitution at residue Thr161 of SEQ ID NO:2; Thr159 of SEQ ID NO:4 orthe equivalent residue in the conserved region of another PL scramblase.41. The method of claim 37, wherein the mutant phospholipid scramblasecomprises a non-functionally equivalent substitution of a cysteineresidue.
 42. The method of claim 41 wherein the cysteine residue to besubstituted is Cys297 of SEQ ID NO:2; Cys295 of SEQ ID NO:4 or theequivalent residue in the conserved region of another PL scramblase. 43.The method of claim 37, wherein the mutant phospholipid scramblasecomprises a non-functionally equivalent substitution of at least one ofthe residues located in the region of Asp273-Asp284 of SEQ ID NO:2;Asp271-Asp282 of SEQ ID NO:4 or the equivalent residues in the conservedregion of another PL scramblase.
 44. The method of claim 37, wherein themutant phospholipid scramblase comprises a non-functionally equivalentsubstitution of at least one residue selected from the group consistingof Asp273, Asp275, Phe277, Ile279, Phe281, and Asp284 of SEQ ID NO:2;Asp271, Asp273, Phe275, Ile277, Phe279, and Asp282 of SEQ ID NO:4 andthe equivalent residues in the conserved region of another PLscramblase.
 45. A method of inhibiting cellular phospholipid scramblasecomprising the steps of delivering to the cell an effective amount of acompound that prevents thioacylation of the scramblase and observing aninhibition of scramblase activity.
 46. The method of claim 45 whereinthe compound is an esterase inhibitor.
 47. The method of claim 45wherein the compound binds with micromolar or greater affinity to thepeptide sequence corresponding to residues Lys290-Cys297 of the PLscramblase SEQ ID NO:2 or the equivalent residues in the conservedregion of another PL scramblase.
 48. The method of claim 47 wherein thecompound is selected from the group consisting of antibody, peptide, ornucleotide.
 49. A method of inhibiting cellular phospholipid scramblasecomprising the steps of delivering to the cell and effective amount of acompound that prevents binding of intracellular Ca²⁺ and observinginhibition of PL scramblase activity.
 50. The method of claim 49 whereinthe compound binds to residues Asp273-Asp284 of SEQ ID NO:2 or theequivalent residues in another PL scramblase.
 51. The method of claim 50selected from the group consisting of antibody, peptide, or nucleotide.52. A method of modifying the activity of cellular phospholipidscramblase comprising the steps of delivering to the cell an effectamount of a compound that prevents phosphorylation of the protein andobserving change in PL scramblase activity.
 53. A method for prolonginggraft survival of transplanted organs and grafts comprising the step ofdelivering a modified PL scramblase to an organ perfusate during invitro organ storage, wherein the modified PL scramblase is modified at aresidue selected from the group consisting of Thr161, Asp273 to Asp284,and Cys297 of SEQ ID NO:2 and the equivalent residues in the conservedregion of another PL scramblase.
 54. A method for prolonging graftsurvival of transplanted organs and grafts comprising the step ofdelivering to an organ perfusate during in vitro organ storage, acompound that prevents postranslational modification of PL scramblase ata residues selected from the group consisting of Thr161, Asp273 toAsp284, and Cys297 of SEQ ID NO:2 and the equivalent residue in theconserved region of another PL scramblase.
 55. A method for prolongingthe in vivo survival of circulating blood cells comprising the step ofpreventing surface exposure of plasma membrane phosphatidylserine on thecirculating blood cells by delivering a modified PL scramblase withinthe blood cells, wherein the modified PL scramblase is modified at aresidue selected from the group consisting of Thr161, Asp273 to Asp284,and Cys297 of SEQ ID NO:2 and the equivalent residues in another PLscramblase.
 56. A method for prolonging the in vivo survival ofcirculating blood cells comprising the step of preventing surfaceexposure of plasma membrane phosphatidylserine on the circulating bloodcells by delivering a compound that prevents postranslationalmodification of PL scramblase at one or more residues wherein thatresidue is selected from the group consisting of Thr161, Asp273 toAsp284, and Cys297 of SEQ ID NO:2 and the equivalent residues in theconserved region of another PL scramblase.
 57. A method for preventingthe procoagulant properties of erythrocytes in sickle cell diseasecomprising the step of delivering a modified PL scramblase in a sicklecell patient, wherein the modified PL scramblase is modified at aresidue selected from the group consisting of Thr161, Asp273 to Asp284,and Cys297 of SEQ ID NO:2 and the equivalent residues in the conservedregion of another PL scramblase.
 58. A method for treating autoimmune,thrombotic, thromboemolic, and inflammatory diseases comprising the stepof treating a patient with a modified PL scramblase, wherein themodified PL scramblase is modified at a residue selected from the groupconsisting of Thr161, Asp273 to Asp284, and Cys297 of SEQ ID NO:2 andthe equivalent residues in the conserved region of another PLscramblase.
 59. A method for treating autoimmune, thrombotic,thromboemolic, and inflammatory diseases comprising the step of treatinga patient with an inhibitor of PL scramblase selected from the group ofcompounds that inhibit postranslational modification of the protein at aresidue selected from the group consisting of Thr161, Asp273 to Asp284,and Cys297 of SEQ ID NO:2 and the equivalent residues in the conservedregion of another PL scramblase.
 60. The method of claim 58, wherein thedisease is selected from disseminated intravascular coagulation,vascular thrombosis, fibrin generation during cardiopulmonary bypassprocedures, rheumatoid arthritis, systemic lupus erythematosus,thrombotic thrombocytopenic purpura, heparin-associated thrombosis, andorgan transplant rejection.